Gag protein is the major structural component of human immunodeficiency virus type 1 (HIV-1) particles and drives virus assembly on cellular membranes. This function of Gag is attributed to its intrinsic self-multimerization ability as well as its interaction with cellular factors such as TSG101 that binds to the PTAP sequence in the p6 region of Gag and promotes HIV-1 budding through recruiting the ESCRT (endosomal sorting complex required for transport). As a result of its essential role in virus assembly, Gag also becomes the target of cellular restriction factors such as APOBEC3G and Trim5alpha. In this study, we report that the fragile X mental retardation protein (FMRP) interacts with HIV-1 Gag and is packaged into virus particles. Although knockdown of FMRP does not markedly affect production of virus particles, infectivity of HIV-1 virions was significantly decreased. Consistent with this observation, overexpression of the wild type FMRP, but not the FMRP mutants that lack the KH1 or the KH2 domains, led to 2- to 3-fold reduction of virus infectivity. Together, these results suggest that FMRP diminishes HIV-1 infectivity through association with viral Gag protein and virus particles.