Stromal‐derived factor‐1 (SDF‐1/CXCL12) and its receptor CXCR4 play crucial roles in leukocyte migration and activation, as well as embryogenesis, angiogenesis, cancer and viral pathogenesis. CXCR4 is one of the major human immunodeficiency virus‐1 (HIV‐1) coreceptors on macrophages. In many tissues macrophages are one of the predominant cell types infected by HIV‐1 and act as a reservoir for persistent infection and viral dissemination. In patients infected by HIV‐1, blood and tissue levels of transforming growth factor‐β1 (TGF‐β1) are increased. The purpose of this study was to evaluate the effects of TGF‐β1 on CXCR4 expression and function in primary human monocyte‐derived macrophages (MDMs) and rat microglia. TGF‐β1 up‐regulated CXCR4 and enhanced SDF‐1α‐stimulated ERK1,2 phosphorylation in these cells. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual‐tropic CXCR4‐using HIV‐1 (D‐X4). In contrast, TGF‐β1 failed to increase CCR5 expression or infection by a CCR5‐using virus in MDMs. Our data demonstrate that TGF‐β1 enhances macrophage responsiveness to SDF‐1α stimulation and susceptibility to HIV‐1 by selectively increasing expression of CXCR4. The results suggest that increased expression of CXCR4 on macrophages may contribute to the emergence of dual‐tropic X4 viral variants at later stages of HIV‐1 infection.