Myocardial fibrosis (MF) is the characteristic pathological feature of various cardiovascular diseases that lead to heart failure (HF) or even fatal outcomes. Alternatively, activated macrophages are involved in the development of fibrosis and tissue remodeling. Although the receptor for advanced glycation end products (RAGE) is involved in MF, its potential role in regulating macrophage function in cardiac fibrosis has not been fully investigated. We aimed to determine the role of macrophage RAGE in transverse aortic constriction (TAC)-induced MF. In this study, we found that RAGE expression was markedly increased in the infiltrated alternatively activated macrophages within mice hearts after TAC. RAGE knockout mice showed less infiltration of alternatively activated macrophages and attenuated cardiac hypertrophy and fibrosis compared to the wild-type mice. Our data suggest that mice with macrophage-specific genetic deletion of RAGE were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload, which led to a decreased proportion of alternatively activated macrophages in heart tissues. Our in vitro experiments demonstrated that RAGE deficiency inhibited the differentiation into alternatively activated macrophages by suppressing autophagy activation. In the co-culture system, in vitro polarization of RAW264.7 macrophages toward an alternatively activated phenotype stimulated the expression of α-smooth muscle actin and collagen in cardiac fibroblasts. However, the knockdown of RAGE and inhibition of autophagy in macrophages showed reduced fibroblast-to-myofibroblast transition (FMT). Collectively, our results suggest that RAGE plays an important role in the recruitment and activation of alternatively activated macrophages by regulating autophagy, which contributes to MF. Thus, blockage of RAGE signaling may be an attractive therapeutic target for the treatment of hypertensive heart disease.© 2023 Federation of American Societies for Experimental Biology.