Human endogenous retrovirus type W (HERV-W) envelope glycoprotein (Env) has recently been reported to induce fusion in cells expressing the RD-114 and type D retrovirus receptor (RDR) and to serve as a functional retroviral envelope protein. In this report, another biological function for HERV-W was demonstrated by testing its ability to protect cells against retroviral infection. Spleen necrosis virus (SNV), a gammaretrovirus was chosen for testing resistance because it uses RDR to enter cells. An HERV-W Env expression plasmid was transfected into canine osteosarcoma cells (D-17), which are permissive for SNV infection. Cell fusion assays were performed to demonstrate biological function of HERV-W Env in D-17 cells. The presence of HERV-W env sequences was confirmed in stably transfected cell clones by using polymerase chain reaction. Viral infectivity assays were performed with SNV and amphotropicMurine leukemia virus (MLV-A) pseudotyped vector viruses to measure titers in D-17 cells expressing HERV-W Env and in negative control cells. The HERV-W Env caused fusion of D-17 cells in culture and greatly reduced infection by SNV vector virus. A 1000- to 10,000-fold decrease in SNV infectivity was observed for D-17 cells expressing HERV-W Env as compared to D-17 cells that were not expressing HERV-W Env. In contrast, infection by MLV-A pseudotyped vector virus was not significantly reduced. Thus, HERV-W Env confers host cell resistance to infection by SNV. This is the first report of a human endogenous retrovirus gene product blocking infection by any exogenous retrovirus.