Objective To screen the substrate protein of ubiquitin-conjugating enzyme E2C (UBE2C) and to explore its role in the progression of hepatocellular carcinoma. Methods An overexpression plasmid of UBE2C was constructed with Halotag and was used to infect the highly metastatic human liver cancer cell line HCCLM3. The UBE2C binding substrate protein was precipitated by Halolink resin and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. UBE2C was overexpressed in human liver cancer cell line Huh-7; the effect on the expression of its substrate protein was detected by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunoflurescence staining, the effect on Wnt signal pathway was detected by cell nucleoplasmic separation combined with Western blotting and TOP flash/FOP flash luciferase reporter assay, and the effect on the expression of Wnt signal pathway downstream genes was further verified by qRT-PCR and Western blotting. Results The mass spectrometry analysis of the differential band of the Halotag coprecipitate obtained by SDS-PAGE and silver staining showed that the substrate protein of UBE2C was β-tubulin. Compared with the control group, overexpression of UBE2C had no effect on the expression of β-tubulin in mRNA level in Huh-7 cells (P>0.05), while it significantly decreased the expression of β-tubulin in protein level (P<0.01). Meanwhile, overexpression of promoted the entry of β-catenin into nucleus (P<0.05), increased the luciferase activity of TOP flash/FOP flash (P<0.01), and enhanced the expression of the Wnt signaling pathway downstream genes (c-Jun N-terminal kinase[JNK], cyclin D1 and matrix metallopeptidase 7) (all P<0.05). Conclusion UBE2C contributes to the progression of hepatocellular carcinoma through decreasing the expUBE2Cression of β-tubulin in hepatocellular carcinoma cells by specifically binding to β-tubulin, activating Wnt signaling pathway and promoting expression of downstream genes.