Objective: To investigate the relations of pre-miR-146a gene expression and its rs2910164 allelic polymorphism with cholangiocarcinoma. Methods: In 70 specimens of cholangiocarcinoma tissues from bile duct cancer patients (cholangiocarcinoma group) and 39 specimens of bile duct tissues from cases with non-neoplastic bile duct diseases (control group), the single nucleotide polymorphism of rs2910164 in pre-miR-146a sequence and pre-miR-146a expression was determined by direct DNA sequencing and the quantitative PCR method, respectively. The relations of different pre-miR-146a genotypes with its expression, and clinicopathological profiles and prognosis of cholangiocarcinoma were analyzed. Results: The genotype distribution in cholangiocarcinoma group was significantly different from that in control group, which in the former showed that the ratio of GG and GC genotypes was significantly higher than that of CC genotype, and the frequency of G allele was significantly higher than that of C allele (both P<0.05). In control group, the pre-miR-146a expression level in cases with GG and GC genotypes was lower than that in cases with CC genotype, but the difference did not reach a statistical significance (P>0.05), while the pre-miR-146a expression level in cholangiocarcinoma tissues was significantly lower than that in the bile duct tissues from control group (P<0.05). Multiple logistic regression analysis showed that GG and GC genotypes were possibly the risk factors for bile duct cancer (P=0.052), and the further factor-stratified analysis showed that GG and GC genotypes were associated with the clinical stage and lymph node metastasis of the patients (both P<0.05). Survival analysis for the cholangiocarcinoma patients demonstrated that the survival rate in cases with GG and GC genotypes was lower than that in cases with CC genotype, but the difference had no statistical significance (P=0.178). Conclusion: The increased frequency of G allele at rs2910164 in pre-miR-146a sequence may be responsible for pre-miR-146a gene under-expression and the risk factor for the occurrence and development of bile duct cancer.