The human immunodeficiency virus type 1 (HIV-1) causes persistent infection in human and induces miR-146a expression in infected cells. miR-146a represses the innate immune response by inhibiting the expression of TRAF6 and IRAK1 genes, thus negatively controls the NF-κB-related cytokines and interferon stimulated genes. Here we reported that lentiviral CRISPR/Cas9 system was highly efficient in introducing mutations in the precursor miR-146a genomic sequences, resulting in a loss of miR-146a expression and function. miR-146a ablation led to increasing cytokines production in LPS-stimulated A549 cells. Moreover, miR-146a knockout in HIV-1 infected MT2 cells markedly increased the expression of cytokines and HIV-1 restriction factors and reversed T cell exhaustion markers expression, thus influencing HIV-1 replication. Our study indicates that lentiviral CRISPR/Cas9-mediated gene editing is an effective approach to abrogate miR-146a expression, which consequently inhibits HIV-1 replication as well as proviral reactivation by enhancing the expression of cytokines and HIV-1 restriction factors.