ABSTRACT Diabetes nephropathy (DN) is characterized by abnormal interactions between kidney‐infiltrating macrophages and glomerular mesangial cells. Recently, a novel cell‐cell communication mediated by exosomes has gained attention. Exosomes are membrane‐bound vesicles that contain a variety of molecules such as proteins, lipids, DNA, mRNA, and microRNAs. Exosomes play an important role in the pathogenesis of DN. In this study, we show that high glucose (HG) led to increased excretion of exosomes from macrophages. Mesangial cells took up exosomes in vitro, which resulted in the activation and proliferation of mesangial cells and the secretion of extracellular matrix and inflammatory cytokines. In addition, C57BL/6 mice injected with exosomes from HG‐treated macrophages showed morphologic and functional changes. We then showed that exosomes from HG‐treated TGF‐β1 knockdown macrophages induced less extracellular matrix and fewer inflammatory factors in mesangial cells compared with vector control. Our findings suggest that TGF‐β1 mRNA in exosomes serves a role between macrophages and mesangial cells by activating the TGF‐β1/ mothers against decapentaplegic homolog 3 pathway.—Zhu, Q.‐J., Zhu, M., Xu, M.‐X., Meng, X.‐M., Wu, Y.‐G. Exosomes from high glucose‐treated macrophages activate glomerular mesangial cells via TGF‐β1/Smad3 pathway in vivo and in vitro. FASEB J. 33, 9279–9290 (2019). www.fasebj.org