It is frequently difficult to determine whether a bile duct stricture is benign or harbors a malignant tumor based on medical history alone. Therefore, cholestasis of unknown etiology requires a thorough diagnostic evaluation to make a definitive diagnosis and choose the best course of treatment for the patient. We report the case of a 42-year-old man who developed cholestasis 6 years after undergoing orthotopic liver transplantation for end-stage liver disease. The bile duct was dilated by interventional endoscopy, and a brush cytology was obtained. Cytology revealed an inflammatory reaction. Three months later, the stricture persisted. We performed a forceps biopsy, and the diagnosis of a cholangiocarcinoma was confirmed histologically. We conclude that a combined application of brush biopsy/forceps biopsy and endoscopic measures is essential in cases of bile duct strictures of unknown etiology. Occasionally, surgical confirmation may be required. In any case, a single brush cytology is insufficient for diagnosis.