BACKGROUND Hepatocellular carcinoma (HCC) is a neoplasm for which the prevalence and mortality rates are very high in Taiwan. The DNA non-homologous end-joining repair gene XRCC6/Ku70 plays an important role in the repair of DNA double-strand breaks (DSBs) induced by both exogenous and endogenous DNA-damaging agents. Defects in overall DSB repair capacity can lead to genomic instability and carcinogenesis. In this study, we investigated the contribution of variant XRCC6 in relation to the risk of HCC, from the levels of DNA, RNA and protein. MATERIALS AND METHODS In this hospital-based case-control study, we collected 298 patients with HCC and 298 cancer-free controls, with frequency matched by age and gender. Firstly, the associations of XRCC6 promoter T-991C (rs5751129), promoter G-57C (rs2267437), promoter A-31G (rs132770), and intron-3 (rs132774) polymorphisms with HCC risk in this Taiwanese population were evaluated. Secondly, 30 HCC tissue samples with variant genotypes were tested to estimate the XRCC6 mRNA expression by real-time quantitative reverse transcription. Finally, the HCC tissue samples of variant genotypes were examined by immunohistochemistry and western blotting to estimate their XRCC6 protein expression levels. RESULTS Compared with the TT genotype, the TC and CC genotypes conferred a significantly increased risk of HCC [adjusted odds ratio (aOR)=2.43 and 3.52, 95% confidence interval (CI)=1.52-4.03 and 1.18-13.36, p=0.0003 and 0.0385, respectively]. The mRNA and protein expression levels in HCC tissues revealed statistically significantly lower XRCC6 mRNA and protein expressions in the HCC samples with TC/CC genotypes compared with those with the TT genotype (p=0.0037 and 0.0003, respectively). CONCLUSION Our multi-approach findings at the DNA, RNA and protein levels suggested that XRCC6 may play an important role in HCC carcinogenesis in the Taiwanese population.