The Serine Incorporator (SERINC) proteins are multipass transmembrane proteins that affect sphingolipid and phosphatidylserine synthesis. Human SERINC5 and SERINC3 were recently shown to possess antiretroviral activity to a number of retroviruses including human immunodeficiency virus (HIV), murine leukemia virus (MLV) and equine infectious anemia virus (EIAV). In the case of MLV, the glycosylated Gag (glyco-Gag) protein was found to counteract SERINC5-mediated restriction in in vitro experiments and that the viral envelope determines virion sensitivity or resistance to SERINC5. However, nothing is known about the in vivo function of SERINC5. Antiretroviral function of a host factor in vitro is not always associated with antiretroviral function in vivo . Using SERINC5-/- mice we generated, we show that mouse SERINC5 (mSERINC5) restriction of MLV infection in vivo is dependent not only on glyco-Gag, but also on the retroviral envelope. Finally, we also examined the in vivo function of the other SERINC gene with known antiretroviral functions, SERINC3. By using SERINC3-/- mice, we found that the murine homologue, mSERINC3, had no antiretroviral role both in vivo and in vitro . This report provides the first data showing that SERINC5 restricts retrovirus infection in vivo and that restriction of retrovirus infectivity in vivo is dependent on both the presence of glyco-Gag and the viral envelope. IMPORTANCE: This study examines for the first time the in vivo function of the Ser ine Inc orporator (SERINC) proteins during retrovirus infection. SERINC3/5 restrict a number of retroviruses including human immunodeficiency virus 1 (HIV-1) and murine leukemia virus (MLV) by blocking their entry into cells. Nevertheless, HIV-1 and MLV encode factors, Nef and glycosylated Gag respectively, that counteract SERINC3/5 in vitro . We recently developed SERINC3 and SERINC5 knockout mice to examine the in vivo function of these genes. We found that SERINC5 potently restricted retrovirus infection in a glycosylated Gag and envelope dependent manner. On the other hand, SERINC3 had no antiviral function. Our findings have implication in the development of therapeutics that target SERINC5 during retrovirus infection.