Low-temperature stress profoundly impairs rice root physiology and reshapes rhizosphere microbial communities. This 2023–2024 study examined its effects on Oryza sativa var. japonica across key growth stages. All treatments significantly suppressed root morphology and function, with the greatest reductions under combined tillering–booting stress (T3), followed by tillering (T1) and booting (T2). Strain DN428 exhibited a stronger cold tolerance than SJ10, with milder declines in root traits. Low-temperature stress elevated soil organic matter and total nitrogen while decreasing available phosphorus and potassium, leading to notable shifts in the microbial community structure and metabolic pathways. Weighted Gene Co-expression Network Analysis identified lacZ, fucK, and rafA in the MEbrown module as potential regulators of varietal cold responses. Mechanistically, yield loss in DN428 was mainly linked to the suppression of microbial gene expression, while in SJ10 it was associated with broader declines in microbial diversity and functional potential. Both varieties experienced yield reductions, accompanied by decreased root activity and nitrogen uptake. These findings underscore the necessity of a “gene–microbe–function” strategy to enhance microbial metabolism and optimize root–soil interactions under cold stress.