Objective: The objective of this study was to evaluate the association of MDR1 gene polymorphisms with susceptibility to hepatocellular carcinoma (HCC). Methods: A total of 689 HCC patients and 680 cancer-free subjects were enrolled. Human MDR1 gene polymorphisms were investigated by created restriction site-polymerase chain reaction (CRS-PCR) and DNA sequencing methods. Multiple logistic regression models were applied to estimate the association between MDR1 gene polymorphisms and susceptibility to HCC. Results: We detected a novel c.4125A>C polymorphism and our findings suggested that this variant was significantly associated with susceptibility to HCC. A significantly increased susceptibility to HCC was noted in the homozygote comparison (CC versus AA: OR=1.621, 95% CI 1.143-2.300, ${chi}^2$=7.4095, P=0.0065), recessive model (CC versus AC+AA: OR=1.625, 95% CI 1.167-2.264, ${chi}^2$=8.3544, P=0.0039) and allele contrast (C versus A: OR=1.185, 95% CI 1.011-1.389, ${chi}^2$=4.4046, P=0.0358). However, no significant increase was observed in the heterozygote comparison (AC versus AA: OR=0.995, 95% CI 0.794-1.248, ${chi}^2$=0.0017, P=0.9672) and dominant model (CC+AC versus AA: OR=1.106, 95% CI 0.894-1.369, ${chi}^2$=0.8560, P=0.3549). Conclusions: These findings suggest that the c.4125A>C polymorphism of the MDR1 gene might contribute to susceptibility to HCC in the Chinese population. Further work will be necessary to clarify the relationship between the c.4125A>C polymorphism and susceptibility to HCC on larger populations of diverse ethnicity.