The latency of human immunodeficiency virus type 1 (HIV-1) in resting primary CD4⁺ T cells is the major barrier for the eradication of the virus in patients on suppressive highly active antiretroviral therapy (HAART). Even with optimal HAART treatment, replication-competent HIV-1 still exists in resting primary CD4⁺ T cells^,,,. Multiple restriction factors that act upon various steps of the viral life cycle could contribute to viral latency. Here we show that cellular microRNAs (miRNAs) potently inhibit HIV-1 production in resting primary CD4⁺ T cells. We have found that the 3′ ends of HIV-1 messenger RNAs are targeted by a cluster of cellular miRNAs including miR-28, miR-125b, miR-150, miR-223 and miR-382, which are enriched in resting CD4⁺ T cells as compared to activated CD4⁺ T cells. Specific inhibitors of these miRNAs substantially counteracted their effects on the target mRNAs, measured either as HIV-1 protein translation in resting CD4⁺ T cells transfected with HIV-1 infectious clones, or as HIV-1 virus production from resting CD4⁺ T cells isolated from HIV-1–infected individuals on suppressive HAART. Our data indicate that cellular miRNAs are pivotal in HIV-1 latency and suggest that manipulation of cellular miRNAs could be a novel approach for purging the HIV-1 reservoir.