Mixed land use (MLU) is recognized as a crucial means for urban planners to facilitate residents’ subjective well-being (SWB). However, prior research has rarely explored the mediating effect of social capital on the MLU-SWB association and has overlooked the possibility that this association may be subject to spatial heterogeneity between residential and workplace neighborhoods. Based on a sample of 1028 survey participants in Shanghai from 2018 to 2019, a structural equation model is used to investigate the associations among MLU, social capital, and SWB in residential and workplace neighborhoods. We find that MLU has a negative and indirect association with SWB by decreasing social capital in residential neighborhoods, while it has a positive and indirect association with SWB by enhancing social capital in workplace neighborhoods with adjustments for other built environmental and sociodemographic attributes. That is, MLU has opposite associations with SWB mediated by social capital. Therefore, policymakers need to adopt different strategies to optimize MLU in residential and workplace neighborhoods to facilitate social capital and SWB.