<p>N-terminal acetylation is a highly abundant protein modification in eukaryotic cells. This modification is catalysed by N-terminal acetyltransferases acting co- or post-translationally. Here, we review the eukaryotic N-terminal acetylation machinery: the enzymes involved and their substrate specificities. We also provide an overview of the impact of N-terminal acetylation, including its effects on protein folding, subcellular targeting, protein complex formation, and protein turnover. In particular, there may be competition between N-terminal acetyltransferases and other enzymes in defining protein fate. At the organismal level, N-terminal acetylation is highly influential, and its impairment was recently linked to cardiac dysfunction and neurodegenerative diseases.</p>