Moloney leukemia virus 10 (MOV10) protein is a superfamily-1 RNA helicase, and it is also a component of the RNA-induced silencing complex. Recent studies have shown that MOV10 plays an active role in the RNA interference pathway. Here, we report that MOV10 inhibits retrovirus replication. When it was overexpressed in viral producer cells, MOV10 was able to reduce the infectivity of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and murine leukemia virus. Conversely, when MOV10 expression was reduced by small interfering RNAs, HIV-1 infectivity was increased. Consistently, silencing of MOV10 expression in a human T cell line enhanced HIV-1 replication. Furthermore, we found that MOV10 interacts with HIV-1 nucleocapsid protein in an RNA-dependent manner and is packaged into virions. It blocks HIV-1 replication at a postentry step. In addition, we also found that HIV-1 could suppress MOV10 protein expression to counteract this cellular resistance. All of these results indicate that MOV10 has a broad antiretroviral activity that can target a wide range of retroviruses, and it could be actively involved in host defense against retroviral infection.