Excess enzyme-mediated protein O-GlcNAcylation is known to occur with diabetes mellitus. A characteristic of diabetic cardiomyopathy is the development of myocardial fibrosis. The role that enhanced protein O-GlcNAcylation plays in modulating the phenotype of cardiac fibroblasts is unknown. To address this issue, rat cardiac fibroblasts (CF) were cultured in normal glucose (NG, 5 mM glucose) or high glucose (HG, 25 mM) media for 48 hours. Results demonstrate that CF cultured in HG have higher levels (~50%) of overall protein O-GlcNAcylation vs. NG cells. Key regulators of collagen synthesis such as TGF-β1, SMADs 2/3, and SMAD 7 protein levels, including those of arginase I and II, were altered, leading to increases in collagen levels. The nuclear transcription factor Sp1 and arginase II evidence excess O-GlcNAcylation in HG cells. Expression in CF of an adenovirus coding for the enzyme N-acetylglucosaminidase which removes O-GlcNAc moieties from proteins, decreased Sp1 and arginase II O-GlcNAcylation and restored HG induced perturbations in CF back to NG levels. These findings may have important pathophysiological implications for the development of diabetes-induced cardiac fibrosis.