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Intercropping, a traditional and worldwide cropping practice, has been considered as a paradigm of sustainable agriculture based on complementary mechanisms among different crop species. Soybean ( Glycine max) is widely relay intercropped about 60 days before maize ( Zea mays) harvest in Southwest China. However, shade caused by tall maize plants may be a limiting factor for soybean growth at a seedling stage. In field research, we studied the ecophysiological responses of two widely cultivated soybean varieties [Gongqiudou494-1 (GQD) and Gongxuan 1 (GX)] to maize shading in a relay intercropping system (RI) compared with monocropped soybean plants (M). Our results showed that soybean seedlings intercropped with maize exhibited significantly downregulated net photosynthetic rate ( P) (−38.3%), transpiration rate (−42.7%), and stomatal conductance (−55.4%) due to low available light. The insignificant changes in intercellular CO concentration and the maximum efficiency of PSII photochemistry suggested that the maize shading-induced depressions in P were probably caused by the deficiency of energy for carbon assimilation. The significantly increased total chlorophyll (Chl) content (+27.4%) and Chl b content (+52.2%), with lowered Chl a/ b ratios (−20.5%) indicated soybean plants adjusted their light-harvesting efficiency under maize shading condition. Biomass and leaf area index (LAI) of seedlings under RI decreased significantly (−78.7 and −71%, respectively) in comparison with M. Correlation analysis indicated the relative reduction in biomass accumulation was caused by the decline in LAI rather than P, it affected negatively the final yields of soybean (32.8%). Cultivar-specific responses to maize shading were observed in respects of LAI, biomass, and grain yield. It indicated that GX might be a better cultivar for relay intercropping with maize in Southwest China. [ABSTRACT FROM AUTHOR]