Previously, we reported that B cell receptor associated protein 31 (BAP31) is a positive regulator on T-cells activation. Helper T cells [cluster of differentiation 4 (CD4) T cells] can regulate macrophage activation in adaptive immune response against pathogens. In this study, we elucidate that M1 and M2 macrophages polarization is significantly suppressed in Lck Cre-BAP31 mice or the co-culture system of CD4 T cells from Lck Cre-BAP31 mice and macrophages from WT mice. It means that BAP31 may affect the regulation of CD4 T cells on macrophages. Further studies suggest that BAP31 deficiency significantly reduce the expressions of T helper 1 (Th1)/ Th2/ Th17/ Th9/ Th22/ Treg cells-related cytokines and transcription factors. The inhibition of macrophages activation caused by BAP31 knockdown is due to the reduction of IFN-γ and IL-4 secreted by Th1 and Th2 cells. BAP31 also affects the levels of early activation markers (CD69 and CD25) of CD4 T cells. Moreover, BAP31 deficiency downregulates the expression of TCRαβ-CD3 complex, and the adaptor proteins p-Zap70, p-Lck, and p-Lat in TCR signaling pathway. These results demonstrate that BAP31 deficiency inhibits TCR/CD3-mediated activation in CD4 T cells and adversely affects macrophages polarization. These findings establish a theoretical foundation for the study of BAP31 in immunotherapy.