Soybeans are the main sources of oil and protein for most of the global population. As the population grows, so does the demand for soybeans. However, drought is a major factor that limits soybean production. Regulating soybean response to drought stress using mepiquat chloride (MC) is a feasible method; however, its mechanism is still unclear. This study used PEG-6000 to simulate drought stress and quantitative proteomic techniques to reveal changes in Heinong44 (HN44) and Heinong65 (HN65) subjected to drought following the application of 100 mg/L of MC. The results showed that SOD in HN44 did not change significantly but decreased by 22.61% in HN65 after MC pretreatment, and MDA content decreased by 22.75% and 21.54% in HN44 and HN65, respectively. Furthermore, MC improved the GSH–ASA cycle and simultaneously promoted the Calvin cycle process to enable the plant to maintain a certain carbon assimilation rate under osmotic stress. In addition, MC upregulated some proteins during gluconeogenesis and starch metabolism and increased soluble sugar content by 8.41% in HN44. MC also reduced ribosomal protein abundance, affecting translation and amino acid metabolism. In summary, MC improved GSH–ASA cycle and Calvin cycle under stress to alleviate oxidative damage and maintain crop growth. Our study is the first to report the mechanism of MC regulation in soybean under osmotic stress, providing new insights for the rational application of MC in soybean.