Protein Nα-terminal acetylation is one of the most common protein modifications in eukaryotic cells, occurring on approximately 80% of soluble human proteins. An increasing number of studies links Nα-terminal acetylation to cell differentiation, cell cycle, cell survival, and cancer. Thus, Nα-terminal acetylation is an essential modification for normal cell function in humans. Still, little is known about the functional role of Nα-terminal acetylation. Recently, the three major human N-acetyltransferase complexes, hNatA, hNatB and hNatC, were identified and characterized. We here summarize the identified N-terminal acetyltransferase complexes in humans, and we review the biological studies on Nα-terminal acetylation in humans and other higher eukaryotes.