Heme metabolism is crucial for the function and longevity of legume nodules, with leghemoglobins (Lbs) serving as the primary heme sink and heme oxygenase (HO) mediating heme degradation. However, the precise functional relationship between HO and Lbs remains unclear. Here, we show that Lotus japonicus HO1 (LjHO1) is strongly induced in early-stage Lb-deficient nodules, but its expression gradually decreases during nodule development. Subcellular localization analysis revealed that LjHO1 is plastid-localized in uninfected cells of lb123 mutant nodules, consistent with its localization in wild-type nodules. Using CRISPR/Cas9, we generated a quadruple ho1lb123 mutant lacking LjHO1 and all three Lb isoforms in L. japonicus. Phenotypic analyses revealed that Lbs deficiency predominantly impairs nitrogen fixation, whereas loss of LjHO1 further reduces nodule formation. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis revealed that loss of Lbs strongly decreased heme accumulation, whereas LjHO1 deficiency slightly increased heme levels in nodules. These results demonstrate that Lbs are essential for heme accumulation and nitrogen fixation, while LjHO1 fine-tunes heme turnover, highlighting their complementary roles in maintaining nodule heme homeostasis and symbiotic efficiency.