Objective: The objective of the present study was to evaluate retrospectively the usefulness of bile cytology to distinguish benign and malignant disease in patients with thickened gallbladder walls using endoscopic transpapillary gallbladder drainage (ETGD) in comparison with computed tomography (CT). Patients and Methods: We reviewed gallbladder diseases in 85 patients visited in our hospital. Twenty-seven cases of gallbladder cancer (GBC) and 58 benign gallbladder diseases were obtained. A pig-tail-type nasobilliary drainage tube was left indwelling in the gallbladder, and washing cytology was performed using this tube. Results: ETGD was performed in 71 of the 85 patients with lesions in thick-walled gallbladders (83.5%) including 22 of the 27 patients with GBC (81.5%) and 49 of the 58 patients with benign gallbladder diseases (84.5%). There were no significant differences in the success rate of ETGD between patients with GBC and those with benign gallbladder diseases CT and cytology using an ETGD tube had 81% and 81% sensitivity, 91% and 83% specificity, and 88% and 82% accuracy, respectively (P > 0.05). Looking only at the 71 successful ETGD cases, CT and ETGD cytology had 82% and 100% sensitivity, 92% and 98% specificity, and 89% and 99% accuracy, respectively. There is statistically significant in the sensitivity and accuracy (P = 0.036 and 0.025, respectively). Four patients with GBC, in whom CT showed benign findings, underwent open cholecystectomy because ETGD cytology revealed malignant findings. In contrast, 4 patients with benign diseases, in whom CT suggested malignant finding, underwent LC because the findings of ETGD cytology suggested benign disease. Positive cytology for GBC in the first, second, third and fourth examination was seen at 50% (11/22), 72% (13/18), 60% (6/10), and 100% (1/1). Accumulative positive rate was 50%, 92%, and 100% in the first, second, and third examination, respectively. Conclusions: ETGD cytology can be conducted following ERC and is useful in differentiating benign and malignant gallbladder diseases. Although success rate of ETGD procedure is limited now, the present study strongly suggests that ETGD cytology is useful in detecting not only lesions that can be discovered by ordinary image diagnosis but also those that are difficult to discover by conventional diagnostic methods.