Abstract Urokinase-type plasminogen activator (uPA), an inducer of macrophage adhesion, inhibits HIV-1 expression in PMA-stimulated, chronically infected U1 cells. We investigated whether uPA-dependent cell adhesion played a role in uPA-dependent inhibition of HIV-1 replication in these cells. Monocyte-derived macrophages (MDM) were generated from monocytes of HIV-infected individuals or from cells of seronegative donors infected acutely in vitro. U1 cells were stimulated in the presence or absence of uPA in standard tissue culture (TC) plates, allowing firm cell adhesion or ultra-low adhesion (ULA) plates. Moreover, U1 cells were also maintained in the presence or absence of vitronectin (VN)-containing sera or serum from VN−/− mice. Virus production was evaluated by RT activity in culture supernatants, whereas cell adhesion was by crystal violet staining and optical microscopy. uPA inhibited HIV replication in MDM and PMA-stimulated U1 cells in TC plates but not in ULA plates. uPA failed to inhibit HIV expression in U1 cells stimulated with IL-6, which induces virus expression but not cell adhesion in TC plates. VN, known to bind to the uPA/uPA receptor complex, was crucial for these adhesion-dependent, inhibitory effects of uPA on HIV expression, in that they were not observed in TC plates in the presence of VN−/− mouse serum. HIV production in control cell cultures was increased significantly in ULA versus TC plates, indicating that macrophage cell adhesion per se curtails HIV replication. In conclusion, uPA inhibits HIV-1 replication in macrophages via up-regulation of cell adhesion to the substrate mediated by VN.