Macrophages, a versatile subset of immune cells, are essential for successful bone repair. Hydrogen sulfide (H2S) is a gasotransmitter associated with tissue development and repair. Emerging evidence demonstrates that H2S is involved in bone formation under physiology condition and bone regeneration under pathology condition. However, whether hydrogen sulfide mediates osteogenesis by influencing macrophages is unknown. Here, we aimed to investigate the effects of hydrogen sulfide on macrophage polarization and the subsequent impact on bone regeneration. In the present study, we found that the H2S-donor GYY4137 stimulated M0/M1 macrophages to express high level of CD-206 and IL-10 but decreased the levels of i-NOS and TNF-α in M1 macrophages. Furthermore, coculture of GYY4137-treated M0 macrophages with pro-osteoblastic MC3T3-E1 cells significantly increased the viability of the MC3T3-E1 cells. Importantly, the formation of mineralized particles in MC3T3-E1 cells was significantly promoted following coculture with IL-4-treated and GYY4137-treated M0 macrophages. Collectively, our study demonstrated that hydrogen sulfide increased macrophages M2 polarization and subsequently promoted bone regeneration.Copyright © 2022 Elsevier B.V. All rights reserved.