N-acetyltransferases are a family of enzymes that catalyze the transfer of the acetyl moiety (COCH3) from acetyl coenzyme A (Acetyl-CoA) to a primary amine of acceptor substrates from small molecules such as aminoglycoside to macromolecules of various proteins. In this study, the substrate selectivity of three N-acetyltransferases falling into different phylogenetic groups was probed against a series of hexosamines and synthetic peptides. GlmA from Clostridium acetobutylicum and RmNag from Rhizomucor miehei, which have been defined as glucosamine N-acetyltransferases, were herein demonstrated to be also capable of acetylating the free amino group on the very first glycine residue of peptide in spite of varied catalytic efficiency. The human recombinant N-acetyltransferase of Naa10p, however, prefers primary amine groups in the peptides as opposed to glucosamine. The varied preference of GlmA, RmNag and Naa10p probably arose from the divergent evolution of these N-acetyltransferases. The expanded knowledge of acceptor specificity would as well facilitate the application of these N-acetyltransferases in the acetylation of hexosamines or peptides.