In addition to acetylation, several acyl modifications at the lysine side chain were identified in vivo including formylation, propionylation, butyrylation, crotonylation and succinylation. Some sirtuin isoforms were reported to preferentially remove “non-acetyl” functional groups, but there are limited data on the acyl specificity of zinc-dependent histone deacetylases. We systematically mapped the acyl specificity of human histone deacetylase 6 (HDAC6) using a panel of carbamoyl phosphate synthetase 1 derived peptides featuring more than 20 acyl modifications. Our data revealed for the first time that in addition to deacetylase, HDAC6 harbors deformylase and depropionylase activities in vitro. Ensuing structure-function and modeling studied provided further insight into lysine deacylation by HDAC6.