The oldest known highly conserved modification of RNA, N4-acetylcytidine, is widely distributed from archaea to eukaryotes and acts as a posttranscriptional chemical modification of RNA, contributing to the correct reading of specific nucleotide sequences during translation, stabilising mRNA and improving transcription efficiency. Yeast Kre33 and human NAT10, the only known authors of ac4C, modify tRNA with the help of the Tan1/THUMPD1 adapter to stabilise its structure. Currently, the mRNA for N4-acetylcytidine (ac4C), catalysed by NAT10 (N-acetyltransferase 10), has been implicated in a variety of human diseases, particularly cancer. This article reviews advances in the study of ac4C modification of RNA and the ac4C-related gene NAT10 in normal physiological cell development, cancer, premature disease and viral infection and discusses its therapeutic promise and future research challenges.