Background and Aims Phosphoserine phosphorylase (PSPH) has been reported to be upregulated in multiple types of cancer and have a promoting effect on tumor growth and metastasis. However, its exact role in hepatocellular carcinoma (HCC) is largely unknown. Therefore, this study was conducted to investigate the expression and action of PSPH in HCC, and to explore the potential mechanism.Methods The expressions of PSPH in HCC and adjacent tissue as well as in different HCC cell lines (HepG2, Huh-7, HCCLM3) and normal hepatic cell line (HL-7702) were detected by Western blot and qRT-PCR, respectively. In HepG2 after overexpression or knock down of PSPH, the changes in proliferation and invasion abilities, and the expressions of proliferation markers CCND1 and Ki-67 were determined by CCK-8, EdU and Transwell invasion assays and Western blot, respectively. Meanwhile, the expressions of autophagy-related proteins LC3-II/LC3-I and p62 as well as invasion-associated protein MMP-9, and nuclear translocation of the p65 submit were detected by Western blot and immunofluorescence staining, respectively.Results The PSPH protein expression was significantly up-regulated in HCC tissue compared with adjacent tissue, and the PSPH gene expressions were significantly increased in each HCC cell line compared with normal hepatic cells (all P<0.05). In HepG2 cells after PSPH overexpression, the proliferation and invasion abilities were significantly enhanced, and the expressions of CCND1 and Ki-67 proteins were significantly up-regulated, with increased LC3-II/LC3-I expression and decreased p62 expression as well as enhanced p65 nuclear translocation and up-regulated MMP-9 expression (all P<0.05); in HepG2 cells after PSPH knockdown, the totally opposite changes in these parameters were observed (all P<0.05). The effects of p65 nuclear translocation promotion and MMP-9 up-regulation by PSPH overexpression were inhibited by NF-κB pathway inhibitor shikonin, and the effects of p65 nuclear translocation inhibition and MMP-9 down-regulation by PSPH knockdown were reversed by NF-κB pathway agonist TNF-α (all P<0.05).Conclusion PSPH expression is increased in HCC and high PSPH expression can strengthen the proliferation and metastasis abilities of HCC cells. The action mechanism may be probably associated with its suppressing autophagy and activating NF-κB/MMP-9 signaling pathway.