IntroductionSoybean is the world’s most important cultivated crop, and drought can affect their growth and, eventually, yields. Foliar application of mepiquat chloride (MC) can potentially alleviate the damage caused by drought stress in plants; however, the mechanism of MC regulation of soybean drought response has not been studied.MethodsThis study investigated the mechanism of soybean drought response regulation by mepiquat chloride in two varieties of soybean, sensitive Heinong 65 (HN65) and drought-tolerant Heinong44 (HN44), under three treatment scenarios, normal, drought stress, and drought stress + MC conditions.Results and discussionMC promoted dry matter accumulation under drought stress, reduced plant height, decreased antioxidant enzyme activity, and significantly decreased malondialdehyde content. The light capture processes, photosystems I and II, were inhibited; however, accumulation and upregulation of several amino acids and flavonoids by MC was observed. Multi-omics joint analysis indicated 2-oxocarboxylic acid metabolism and isoflavone biosynthetic pathways to be the core pathways by which MC regulated soybean drought response. Candidate genes such asLOC100816177, SOMT-2, LOC100784120, LOC100797504, LOC100794610, andLOC100819853were identified to be crucial for the drought resistance of soybeans. Finally, a model was constructed to systematically describe the regulatory mechanism of MC application in soybean under drought stress. This study fills the research gap of MC in the field of soybean resistance.