MicroRNAs (miRNAs) are important host molecules involved in human immunodeficiency virus type 1 (HIV-1) infection. Antiretroviral therapy (ART) can affect the miRNA expression profile, but differentially expressed miRNAs still remain to be identified. In this study, we used gene chips to analyze miRNA expression profiles in peripheral blood mononuclear cells from ART-naive HIV-1 patients and those receiving ART, as well as from uninfected individuals. We measured differences in miRNA expression by quantitative polymerase chain reaction (qPCR) in an expanded sample. We found significant differences in the expression of has-miR-191-5p among the three groups (P < 0.05). Furthermore, we showed that hsa-miR-191-5p has an inhibitory effect on HIV-1 replication in cell models in vitro. We identified CCR1 and NUP50 as target molecules of hsa-miR-191-5p and found that hsa-miR-191-5p inhibits the expression of CCR1 and NUP50. Knockdown of NUP50 resulted in significant inhibition of HIV-1 replication. In summary, our research shows that hsa-miR-191-5p expression is reduced in HIV-1-infected patients and acts an inhibitor of HIV-1 infection via a mechanism that may involve targeted repression of NUP50 expression.