Cold stress is the major abiotic factor limiting crop productivity. However, compared to model plant Arabidopsis thaliana, crucial genes and underlying molecular mechanisms involved in soybean cold stress remain underexplored. Here, we investigate two national soybean cultivars, HH43 and HX3, bred from northeast and southwest regions of China, which exhibit significant differences in cold tolerance. Morphological and biochemical examinations show that, compared to HH43, HX3 exhibits delayed wilting, reduced oxidative damage, and elevated antioxidant enzyme activities under low temperature (4°C). Transcriptomic analyses at different time courses upon 4°C show distinct patterns, with HH43 having a rapid gene response and HX3 exhibiting a gradual increase. Particularly, we identify MEblue and MEgreen modules related to cold stress and construct their GENIE3 networks. Key TFs such as CAMTA1/A2 , WRKY33/40 , ATAF1 , ERF72, bZIP29 and their potential targets were identified, which likely contribute to the cold tolerance differences between HH43 and HX3. Moreover, proteomic analyses reveal a broader and more sustained protein upregulation associated with maintaining metabolic activity and cellular homeostasis in cold-tolerant HX3, while limited proteomic response was observed in cold-sensitive HH43 and degradation of certain early response proteins. These findings provide substantial resources for further functional research and breeding cold-tolerant soybean cultivars.