The goal of this study was to address the role of ATP-citrate lyase (ACL), an enzyme that converts citrate to acetyl-CoA, in high glucose (HG)-induced histone acetylation and pro-fibrotic gene expression. Our recent ChIP-Seq studies have demonstrated that HG induces genome-wide histone hyperacetylation in mesangial cells (MCs). Here we showed that exposure of MCs to HG markedly increased histone acetylation at the H3K9/14 and H3K18 marks and induced the expression of potent pro-fibrotic factors TGF-β1, TGF-β3 and CTGF. The induction of these pro-fibrotic factors was further enhanced by histone deacetylase inhibitor but suppressed by histone acetyl-transferase inhibitor, confirming the importance of histone acetylation in this regulation. Interestingly, HG not only up-regulated ACL expression but also promoted ACL nuclear translocation, evidenced by increased ACL concentration and activity in the nuclear extracts. Consistent with this observation, transfection of MCs with a plasmid carrying GFP-ACL fusion protein led to GFP nuclear accumulation when cultured in HG condition. Silencing ACL with siRNAs alleviated HG-induced histone hyperacetylation as well as up-regulation of TGF-β1, TGF-β3, CTGF and extracellular matrix (ECM) proteins fibronectin and collagen type IV, whereas ACL overexpression further enhanced HG induction of histone acetylation as well as these pro-fibrotic factors and ECM proteins. Collectively, these observations demonstrate that HG promotes ACL expression and translocation into the nucleus, where ACL converts citrate to acetyl-CoA to provide the substrate for histone acetylation, leading to up-regulation of fibrogenic genes. Therefore, ACL plays a critical role in epigenetic regulation of diabetic renal fibrosis.