Co-translational N-terminal protein acetylation regulates many protein functions including degradation， folding， interprotein interactions， and targeting. Human NatA (hNatA)， one of six conserved metazoan N-terminal acetyltransferases， contains Naa10 catalytic and Naa15 auxiliary subunits， and associates with the intrinsically disordered Huntingtin yeast two-hybrid protein K (HYPK). We report on the crystal structures of hNatA and hNatA/HYPK， and associated biochemical and enzymatic analyses. We demonstrate that hNatA contains unique features: a stabilizing inositol hexaphosphate (IP) molecule and a metazoan-specific Naa15 domain that mediates high-affinity HYPK binding. We find that HYPK harbors intrinsic hNatA-specific inhibitory activity through a bipartite structure: a ubiquitin-associated domain that binds a hNaa15 metazoan-specific region and an N-terminal loop-helix region that distorts the hNaa10 active site. We show that HYPK binding blocks hNaa50 targeting to hNatA， likely limiting Naa50 ribosome localization in vivo. These studies provide a model for metazoan NAT activity and HYPK regulation of N-terminal acetylation.