The antiviral activity of host factor apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G, A3G) and its degradation mediated by human immunodeficiency virus type 1 (HIV-1) Vif protein are important topics. Although accumulating evidence indicates the importance of deubiquitination enzymes (DUBs) in innate immunity, it is unknown if they participate in A3G stability. Here, we found that USP49 directly interacts with A3G and efficiently removes ubiquitin, consequently increasing A3G protein expression and significantly enhancing its anti-HIV-1 activity. Unexpectedly, A3G degradation was also mediated by a Vif- and cullin-ring-independent pathway, which was effectively counteracted by USP49. Furthermore, clinical data suggested that USP49 is correlated with A3G protein expression and hypermutations in Vif-positive proviruses, and inversely with the intact provirus ratio in the HIV-1 latent reservoir. Our studies demonstrated a mechanism to effectively stabilize A3G expression, which could comprise a target to control HIV-1 infection and eradicate the latent reservoir.