The internal N⁶-methyladenosine (m⁶A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m⁶A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1-3) bind to m⁶A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1-3 proteins recognize m⁶A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4⁺ T-cells. We further mapped the YTHDF1-3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1-3 in cells had the opposite effects. Moreover, silencing the m⁶A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m⁶A erasers increased Gag expression. Our findings suggest an important role of m⁶A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis.