Epitranscriptomic RNA modifications, including methylation of adenine and cytidine residues, are now recognized as key regulators of both cellular and viral mRNA function. Moreover, acetylation of the N⁴ position of cytidine (ac4C) was recently reported to increase the translation and stability of cellular mRNAs. Here, we show that ac4C and N-acetyltransferase 10 (NAT10), the enzyme that adds ac4C to RNAs, have been subverted by human immunodeficiency virus 1 (HIV-1) to increase viral gene expression. HIV-1 transcripts are modified with ac4C at multiple discrete sites, and silent mutagenesis of these ac4C sites led to decreased HIV-1 gene expression. Similarly, loss of ac4C from viral transcripts due to depletion of NAT10 inhibited HIV-1 replication by reducing viral RNA stability. Interestingly, the NAT10 inhibitor remodelin could inhibit HIV-1 replication at concentrations that have no effect on cell viability, thus identifying ac4C addition as a potential target for antiviral drug development.