Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin‐derived oligosaccharides (>8 sugars) and N‐desulfated or O‐desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive‐like inhibitor causing an ∼50‐fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an ∼50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild‐type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation. J. Cell. Biochem. 105: 108–120, 2008. © 2008 Wiley‐Liss, Inc.