Lactate accumulation is an important metabolic feature of hepatocellular carcinoma and may regulate tumor progression through lactate-dependent post-translational modifications. In this study, we investigated whether lactate-induced protein lactylation contributes to the regulation of phosphoglycerate kinase 1 (PGK1) in hepatocellular carcinoma cells. HCC cells were treated with lactate, and endogenous PGK1 was immunoprecipitated for mass spectrometry analysis. LC-MS/MS analysis identified lysine lactylation of PGK1, with K131 detected as a lactylated residue.