Waxy sorghum–soybean intercropping is a sustainable and intensive farming system in southwest China. However, there is limited knowledge about the effects of intercropped soybean combined with nitrogen application on nitrogen uptake and utilization in waxy sorghum. A two-year (2023 and 2024) field experiment was carried out using a randomized complete block design with three planting patterns and three nitrogen application rates to explore the responses of grain yield formation and nitrogen uptake, accumulation, transportation, metabolism physiology, and utilization of waxy sorghum for intercropped soybean combined with nitrogen application. Planting patterns included sole cropped waxy sorghum (SCW), sole cropped soybean (SCS), and waxy sorghum intercropped with soybean (WSI), and nitrogen application rates included zero nitrogen (N0), medium nitrogen (N1), and high nitrogen (N2). Results showed that the dry matter accumulation amount, nitrogen content, nitrogen accumulation amount, nitrogen transportation amount, nitrogen transportation rate, contribution rate of nitrogen transportation to grains, nitrogen metabolizing enzymes activities (including nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthetase, glutamate dehydrogenase, and glutamic-pyruvic transaminase), and active substances contents (including soluble sugar, soluble protein, and free amino acid) in various organs of waxy sorghum among planting patterns and nitrogen application rates were in the order of WSI > SCW and N1 > N2 > N0, respectively. In addition, the nitrogen uptake efficiency, nitrogen agronomy efficiency, nitrogen apparent efficiency, nitrogen recovery efficiency, nitrogen partial factor productivity, and nitrogen contribution rate of waxy sorghum among planting patterns and nitrogen application rates were in the sequence of WSI > SCW and N1 > N2, respectively. The changes in above traits resulted in the WSI-N1 treatment obtaining the highest grain yield (6020.66 kg ha⁻¹ in 2023 and 6159.81 kg ha⁻¹ in 2024), grain weight per spike (65.22 g in 2023 and 64.51 g in 2024), 1000-grain weight (23.14 g in 2023 and 23.18 g in 2024) of waxy sorghum, and land equivalent ratio (1.41 in 2023 and 1.44 in 2024). Overall, waxy sorghum intercropped with soybean combined with medium nitrogen application (220 kg ha⁻¹ for waxy sorghum and 18 kg ha⁻¹ for soybean) can help enhance the nitrogen uptake and utilization of waxy sorghum by improving nitrogen metabolizing enzymes’ activities and active substances’ contents, thereby promoting its productivity.

Sorghum [Sorghum bicolor (L.) Moench] production faces persistent challenges from low nitrogen-use efficiency (NUE) under the intensive cultivation systems of Southwest China. To elucidate the physiological determinants of genotypic variation in NUE, a two-site field experiment was conducted in Guiyang and Anshun using a high-NUE waxy sorghum cultivar (HLF) and a low-NUE waxy sorghum cultivar (QV9) under two nitrogen levels: no nitrogen application (BSN, 0 kg N ha⁻¹) and conventional nitrogen application (SN, 150 kg N ha⁻¹). Results showed that grain yield (Yield) and NUtE responded significantly to genotype (G), nitrogen application rate (N), and G × N effects across both sites. For NUpE, the main effect of N was significant at Anshun but not at Guiyang, whereas genotypic differences were consistent across both sites. Under BSN treatment, HLF exhibited a modest 11.04% yield reduction relative to SN, whereas QV9 suffered a 24.31% decline. Across both nitrogen levels and sites, HLF consistently outperformed QV9 in NUpE and NUtE, with NUpE reaching 1.58–1.98 times that of QV9. Nitrogen application significantly enhanced leaf area index (LAI) and SPAD values, and HLF maintained higher canopy indices at post-anthesis compared to QV9. Total dry matter and nitrogen accumulation (TDMA and TNA), post-anthesis dry matter and nitrogen accumulation (DMAP and NAP), and proportion of post-anthesis dry matter and nitrogen accumulation (DMAPP and NAPP) of HLF were higher than QV9. Although the BSN-HLF produced higher dry matter translocation amounts than BSN-QV9, HLF exhibited lower dry matter and nitrogen translocation rates (DMTR and NTR) and contribution rates of dry matter and nitrogen to grains (DMTCR and NTCR). At the enzymatic level, HLF sustained significantly elevated activities of nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT) and maintained elevated antioxidant enzyme activities (SOD and POD) at both anthesis and maturity stages under BSN treatment. Concurrently, HLF accumulated higher concentrations of osmolytes (proline and soluble sugars), while maintaining reduced malondialdehyde (MDA) content, indicative of strengthened cellular homeostasis under BSN treatment. Mantel tests further revealed that DMAPP, GS, GOGAT, POD, SSC, and MDA were commonly and significantly linked to both NUE and yield. Overall, HLF maintained higher NUE and yield stability under low nitrogen, which may be associated with its sustained nitrogen metabolic enzyme activities, optimized canopy performance, and enhanced antioxidant and osmotic adjustment capacities.