Dysregulated metabolism is an emerging hallmark of cancer development, and upregulated lipid synthesis is one of the important tumor metabolic features. However, lipolysis may also contribute to cancer pathogenesis by altering free fatty acid (FFA) metabolism. In the present study, we investigated the importance of the lipolytic enzyme acyl-CoA thioesterase 8 (ACOT8) in hepatocellular carcinoma (HCC) development. Bioinformatic analysis of published microarrays regarding clinical specimens revealed that both ACOT8 gene copy number and mRNA expression were increased in HCC tissues when compared to these variables in non-tumor tissues. ACOT8 silencing with specific shRNA stably expressed in Huh7 and Hep3B HCC cell lines showed that ACOT8 protein expression and overall thioesterase activity were reduced following ACOT8 knockdown. In vitro tumorigenic assays revealed that ACOT8 knockdown inhibited anchorage-dependent and ‑independent growth of HCC cell lines. This growth inhibition was partially rescued by addition of the FFA, myristic acid, indicating the importance of FFA in cancer metabolism. In summary, lipolytic enzyme ACOT8 is frequently upregulated in HCC clinical specimens. More importantly, ACOT8 silencing leads to inhibition of cell growth in HCC in vitro.