Liver fibrosis results from excessive extracellular matrix (ECM) deposition following chronic hepatocyte injury. Hepatic stellate cell (HSC) activation is central to fibrogenesis; however, the regulatory mechanisms controlling this process remain unclear. Protein acetylation, regulated by histone acetyltransferases and deacetylases (HDACs), modulates gene expression and protein stability. To date, no studies have explored acetylation dynamics during HSC activation using proteomic approaches. In this study, we investigated the role of acetylation in HSC activation and liver fibrosis. Proteomic profiling of primary mouse HSCs was performed to assess global protein acetylation and the expression of HDACs and SIRTs during HSC activation. Activated HSCs exhibited reduced global protein acetylation, increased HDAC2/3/6 expression, and decreased SIRTs expression. Selective inhibition of HDAC6, but not HDAC2/3, suppressed TGF-β-induced HSC activation. TGF-β increased HDAC6 expression via Smad3-dependent transcription, and HDAC6 promoted fibrogenic gene expression through Smad activation. Proteomic screening identified acetyl-CoA acetyltransferase 1 (ACAT1) as a novel HDAC6 substrate; HDAC6 overexpression enhanced ACAT1 binding and reduced its acetylation. In vivo, pharmacological inhibition of HDAC6 lowered ALT and AST levels and improved histological changes in fibrotic mice. Our results suggest that selective HDAC6 inhibition may represent a promising therapeutic strategy for treating liver fibrosis.