Intrahepatic cholangiocarcinoma (iCCA) has a poor prognosis and limited treatment options. Aprepitant, a selective NK-1R antagonist, is capable of inhibiting the growth of various tumors in vitro and in vivo. To investigate the cytotoxic effects of aprepitant on the intrahepatic cholangiocarcinoma, we confirmed the expression of SP/NK-1R in clinical samples of iCCA by immunohistochemistry. MTT test showed that aprepitant could suppress cell viability of RBE and HCCC-9810. Meanwhile, aprepitant induced mitochondria-dependent apoptosis through ROS/JNK pathway in a dose-dependent manner. Additionally, anti-apoptotic drug z-VAD-fmk pretreatment could partly improve the decrease of cell viability induced by aprepitant, while ROS scavenger, NAC could cancel the cytotoxic effects of aprepitant in vitro. Therefore, these results indicated that autophagy is another downstream process of aprepitant-induced ROS accumulation. Changes in the expression of autophagy-related proteins and number of autophagosomes were detected by western blotting and electron microscopy, respectively. Furthermore, we observed the dynamic changes of autophagosome in RBE and HCCC-9810 treated with aprepitant by using mRFP-eGFP-LC3 adenovirus transfection. Moreover, aprepitant significantly inhibited the growth of iCCA xenografts. These findings reveal NK-1R as an potential therapeutic target against iCCA and highlight the anti-cancer ability of NK-1R antagonist aprepitant in iCCA.