The interaction between immune regulatory cells, such as regulatory B cells (Breg) and pro-resolving macrophages (M2 macrophages), plays an important role in the restoration of immune homeostasis during inflammation. PD-L1 is one of the effector molecules that mediates the immune regulation function of M2 macrophages. The activation of PD-L1/PD-1 signaling promotes the differentiation of Breg. Previous studies have shown that Breg promoted M2 macrophage polarization and enhanced their function, but little is known about the regulatory function of M2 macrophages on Breg differentiation. This study aims to determine the effect of M2 macrophages on Breg induction and the potential mechanism in vitro. Bone-marrow-derived macrophages were isolated from wild-type (WT) mice and polarized into M2 using IL-4/IL-13. To investigate the role of PD-L1/PD-1 in M2 macrophage-induced Breg differentiation, spleen B cells were isolated from WT or PD-1 knockout (KO) mice and co-cultured with either na & iuml;ve (M0) or M2 macrophages for 48 h with or without trans-well inserts. The expression of IL-10, IL-35, and TGF-beta 1 in B cells was evaluated by flow cytometry and immunofluorescence staining. Recombinant PD-L1 was used to stimulate activated B cells, followed by the detection of IL-35 and TGF-beta 1. The results show that there was no significant difference in IL-10 expression among all groups. However, IL-35 and TGF-beta 1 expression in B cells was significantly increased in the M2+B, but not in M0+B, compared to B cells alone. Notably, such increases were diminished when M2 and B cells were separated by trans-well inserts. IL-35 expression was not significantly changed when B cells from PD-1 KO mice were co-cultured with M2 compared to the control. However, TGF-beta 1 expression was significantly increased when PD-1 KO B cells were co-cultured with M2 compared to the control. IL-35 expression in activated B cells was increased upon stimulation with PD-L1. However, TGF-beta 1 expression in activated B cells was increased regardless of the PD-L1 availability. This study demonstrates that pro-resolving macrophage-induced IL-35+ but not TGF-beta 1+ regulatory B cell activation requires the PD-L1/PD-1 pathway.