Gallbladder cancer (GBC) is one of the most common malignancy of the biliary tract characterized by its high chemoresistant tendency. Although great progresses have been made in recent decades for treating many cancers with anticancer drugs, effective therapeutics methods for anti-GBC are still lacking. Therefore, investigations into identifying the mechanisms underlying the drug resistance of GBC are greatly needed. In this study, we show that miR-218-5p plays a critical role in gemcitabine resistance of GBC. miR-218-5p levels were significantly lower in GBC than adjacent non-cancer tissues, and which were also associated with patient prognosis. While miR-218-5p overexpression abrogated gemcitabine resistance of GBC cells, silencing of which exhibited the opposite effects. Via six microRNA targets prediction algorithms, we found that PRKCE is a potential target of miR-218-5p. Moreover, miR-218-5p overexpression repressed the luciferase activity of reporter constructs containing 3'-UTR of PRKCE and also reduced PRKCE expression. Further studies revealed that miR-218-5p promotes sensitivity of gemcitabine by abolishing PRKCE-induced upregulation of MDR1/P-gp. Taken together, our results imply that an intimate correlation between miR-218-5p and PRKCE/MDR1 axis abnormal expression is a key determinant of gemcitabine tolerance, and suggest a novel miR-218-5p-based clinical intervention target for GBC patients.