Objective: To investigate the risk factors associated with gallbladder cancer, and provide a theoretical basis for the prevention and treatment of this condition. Methods: Using a case-control design, 316 patients admitted and diagnosed as gallbladder cancer from January 2009 to December 2017 in Xiangya Hospital of Central South University and 316 cases admitted for other diseases with similar age distribution and sex ratio to the former during the same period were selected. The risk factors associated with the occurrence of gallbladder cancer was analyzed. Results: Of the 316 patients with gallbladder cancer, the mean age was (60.2±10.6) years, cases over 50 years of age accounted for 82.28%, the male to female ratio was 1:1.95, and 156 cases (49.4%) had concomitant gallstones, of whom 30 cases were gallbladder filling type stones. Univariate and multivariate analysis showed that gallstone was the only risk factor for gallbladder cancer (OR=6.72, 95% CI=4.52–10.02, P<0.01). The number of cases with gallbladder cancer and concomitant gallstones showed a first rising and then declining trend during the studied period, and its proportion accounting for the total number of cases of gallbladder cancer each year did not significantly change; the proportion of cases with concomitant gallstones in female patients was higher than that in male patients (P<0.01). The proportions of gallbladder cancer patients with concomitant gallstones who did not undergo radical surgery and had TNM stage IIIB and IV diseases were higher than those of gallbladder cancer patients without gallstones (both P<0.05). The relative risks to gallbladder cancer were increased in patients with large-diameter gallstones and gallbladder filling type stones (both P<0.05). Conclusion: Gallstones may be one of the major risk factors for gallbladder cancer. The risk for gallbladder cancer is increased with the increase of the diameter and number of the gallstones. The gallbladder cancer patients with concomitant gallstones always have a relatively late stage. Timely surgical treatment is recommended for those with types of stones that are relatively possible to develop gallbladder cancer.