BACKGROUND Serine hydroxymethyltransferase 2 (SHMT2) is a key enzyme in one-carbon cell metabolism, including in liver cancer. However, the associations between SHMT2 expression at the gene and protein level and prognosis in patients with hepatocellular carcinoma (HCC) remains unknown. This study aimed to investigate the expression levels of SHMT2 in tumor tissue samples from patients with HCC and clinical outcome and the effects of silencing the expression of the SHMT2 gene in HepG2 cells. MATERIAL AND METHODS Expression levels of SHMT2 were evaluated in 144 cases of HCC using immunohistochemistry and correlated with clinicopathological factors using the chi-squared (χ²) test. The prognostic significance of SHMT2 expression was analyzed by univariate analysis and multivariate analysis. Twenty pairs of HCC tissue and adjacent normal liver tissue were compared for SHMT2 expression levels using quantitative reverse transcription polymerase chain reaction (qRT-PCR). HepG2 cells underwent SHMT2 gene silencing and MTT and transwell assays investigated cell proliferation and migration. Western blot was used to detect the expression of markers of epithelial-mesenchymal transition (EMT). RESULTS Expression levels of SHMT2 in HCC tissues were significantly correlated with tumor grade and hepatitis B virus (HBV) infection, and increased expression was an independent negative prognostic factor in patients with HCC (P=0.003). Increased expression of the SHMT2 gene promoted the proliferation and migration of the HepG2 HCC cell line. CONCLUSIONS Increased expression of SHMT2 was a negative prognostic biomarker in patients with HCC. Expression of the SHMT2 gene promoted the proliferation and migration of HepG2 HCC cells.