Objective To construct the implantation model of human hilar cholangiocarcinoma in situ in nude mice and to analyze the nerve infiltration of cholangiocarcinoma cells in these tumors. Methods After anesthesia, nude mice were given laparotomy. Then, cultured human hilar cholangiocarcinoma cell line QBC939 was injected in the interaction between the hilar bile duct and portal vein of nude mice. Ultrasonic assay was used to determine the location and the status of the tumor. Two weeks after implantation, nude mice were sacrificed for the anatomical and pathological observation. In addition, S100 protein was detected using immunohistochemistry to determine the status of nerve infiltration in the tumor specimens. Results All nude mice implanted with QBC939 cells developed tumors in situ as revealed by Ultrasonic assay and anatomy. Tumor cells were highly aggressive and invaded into the surrounding organs and lymph node. 8 of 10 (80%) tumor specimens had nerve infiltration. Conclusion The implantation model of human hilar cholangiocarcinoma in situ with high ratio of nerve infiltration in nude mice serves as ideal platinum for exploring the mechanism of the development and progression of hilar cholangiocarcinoma and determining the effective treatment strategies.