Envelope (env) proteins of certain endogenous retroviruses (ERVs) participate in various pathophysiological processes. In this study, we characterized pathophysiologic properties of two murine leukemia virus-type ERV (MuLV-ERV) env genes cloned from the ovary of C57BL/6J mice. The two env genes (named ENV(OV1) and ENV(OV2)), with 1,926 bp coding region, originated from two MuLV-ERV loci on chromosomes 8 and 18, respectively. ENV(OV1) and ENV(OV2) were ~75 kDa and predominantly expressed on the cell membrane. They were capable of producing pseudotype murine leukemia virus virions. Tropism trait and infectivity of ENV(OV2) were similar to the polytropic env; however, ENV(OV1) had very low level of infectivity. Overexpression of ENV(OV2), but not ENV(OV1), exerted cytotoxic effects and induced expression of COX-2, IL-1β, IL-6, and iNOS. These findings suggest that the ENV(OV1) and ENV(OV2) are capable of serving as an env protein for virion assembly, and they exert differential cytotoxicity and modulation of inflammatory mediators.