Significance N-terminal acetylation performed by N-terminal acetyltransferases (NATs) is a common protein modification in human cells. A unique NAT, NAA80, was recently found to control actin N-terminal acetylation and cytoskeletal dynamics. In this study, we developed potent and specific bisubstrate inhibitors against NAA80 and determined the crystal structure of NAA80 in complex with an inhibitor mimicking the β-actin N terminus, thus revealing molecular determinants for the substrate specificity and selective inhibition of NAA80. A yeast model uncovered how a cellular determinant, the NatB enzyme, acts to restrict the number of in vivo NAA80 substrates relative to the broader intrinsic capacity of NAA80. Our data provide a starting point for further development of inhibitors for the regulation of actin and cytoskeletal functions.