Protein lysine acetylation is a vital post-translational modification (PTM) that plays important roles in biological processes and human diseases. However, its potential roles in hepatocellular carcinoma (HCC) development remain largely unknown. Here we performed a quantitative acetylome analysis of tumor and normal liver tissues from HCC patients. Overall, we identified 792 lysine acetylation sites in 415 proteins, and almost half of their acetylation levels were significantly changed in HCC tumor tissues. The acetylated proteins mainly consisted of metabolic enzymes, which was consistent with the subcellular location analysis that they were primarily located in mitochondria and cytoplasm. Consistently, Bioinformatics analysis showed that differently acetylated proteins were mainly involved in metabolic pathways, such as glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid oxidation, and glutamine metabolism. Then we selected two down-regulated enzymes and verified their acetylation levels in HCC liver tissues. In addition, transcription factors and proteins associated with oxidative stress were identified with aberrant acetylation levels in HCC tumor tissues. Our findings illustrate abundant lysine acetylation sites in HCC liver tissues, which provides insight into the role of lysine acetylation in HCC development, and further contributes to possible implications for its use in diagnose and therapy in the future.