Leishmania presents a complex life cycle that involves both invertebrate and vertebrate hosts. By regulating gene expression, protein synthesis, and metabolism, the parasite can adapt to various environmental conditions. This regulation occurs mainly at the post-transcriptional level and may involve epitranscriptomic modifications of RNAs. Recent studies have shown that mRNAs in humans undergo a modification known as N4-acetylcytidine (ac4C) catalyzed by the enzyme N-acetyltransferase (NAT10), impacting mRNAs stability and translation. Here, we characterized the NAT10 homologue of L. mexicana, finding that the enzyme exhibits all the conserved acetyltransferase domains although failed to functionally complement the Kre33 mutant in Saccharomyces cerevisiae. We also discovered that LmexNAT10 is nuclear, and seems essential, as evidenced by unsuccessful attempts to obtain null mutant parasites. Phenotypic characterization of single-knockout parasites revealed that LmexNAT10 affects the multiplication of procyclic forms and the promastigote-amastigote differentiation. Additionally, in vivo infection studies using the invertebrate vector Lutzomyia longipalpis showed a delay in the parasite differentiation into metacyclics. Finally, we observed changes in the cell cycle progression and protein synthesis in the mutant parasites. Together, these results suggest that LmexNAT10 might be important for parasite differentiation, potentially by regulating ac4C levels.