Liver fibrosis is driven by excessive extracellular matrix deposition following chronic hepatocyte injury, with hepatic stellate cell (HSC) activation playing a central role. However, the mechanisms regulating this process remain unclear. Protein acetylation, controlled by histone acetyltransferases and deacetylases (HDACs), influences gene expression and protein stability, yet its role in HSC activation has not been fully explored. Here, proteomic analysis of primary mouse HSCs revealed reduced global protein acetylation during activation, along with increased HDAC2/3/6 and decreased SIRT expression. Selective inhibition of HDAC6, but not HDAC2/3, suppressed TGF-β-induced HSC activation. Mechanistically, TGF-β upregulated HDAC6 via Smad3, and HDAC6 enhanced fibrogenic gene expression through Smad signaling. Proteomics identified ACAT1 as a novel HDAC6 substrate, with HDAC6 reducing its acetylation. In vivo, HDAC6 inhibition improved liver injury markers and histology in fibrotic mice, suggesting therapeutic potential. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [No. RS-2023-00222390 and RS- 2025-00517344 (S.H.K)].