Objective: To investigate the TUSC3 gene expression in hepatocellular carcinoma (HCC) and its clinical significance. Methods: The paraffin-embedded tissue specimens and clinicopathologic data of 92 patients with HCC confirmed by postoperative pathology were collected, and the TUSC3 expressions in HCC and the adjacent tissues were detected by immunohistochemical staining and the relations of TUSC3 expression with clinicopathologic factors of the patients were analyzed. The mRNA and protein expressions of TUSC3 in 20 paired frozen tissue samples of HCC and adjacent tissues were determined by qRT-PCR and Western blot analysis, respectively. Results: Results of immunohistochemical staining for the 92 paraffin-embedded tissue specimens showed TUSC3 expression down-regulated in 56 cases (60.9%) in HCC tissues, and only in 33 cases (35.9%) in adjacent tissues, and the difference was statistically significant (P<0.001); low TUSC3 expression was significantly related to Edmondson grade (P=0.008), TNM stage (P=0.031) and tumor size (P=0.020). Results of qRT-PCR and Western blot analysis of the 20 paired fresh samples showed that the relative expression levels of both TUSC3 mRNA [(0.99±1.46) vs. (1.96±2.18)] and protein [(0.49±0.35) vs. (1.04±0.43)] in HCC tissue were significantly lower than those in adjacent tissue (both P<0.05). Conclusion: The TUSC3 gene expression is down-regulated in HCC tissue and the low TUSC3 expression may be closely related to the malignant process of HCC.