Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of thebile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crudeethanolic extract of ginger (Zingiber officinale Roscoe) against CCA. Cytotoxic activity against a CCA cell line(CL-6) was assessed by calcein-AM and Hoechst 33342 assays and anti-oxidant activity was evaluated using theDPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction ofgenes that may be involved in the resistance of CCA to anticancer drugs (MDR1, MRP1, MRP2, and MRP3)was examined by real-time PCR. To investigate anti-CCA activity in vivo, a total of 80 OV and nitrosamine (OV/DMN)-induced CCA hamsters were fed with the ginger extract at doses of 1000, 3000, and 5000 mg/kg bodyweight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group werefed with 5-fluorouracil (positive control) or distilled water (untreated control). Median IC50 (concentration thatinhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract ofginger were 10.95, 53.15, and 27.86 μg/ml, respectively. More than ten DNA fragments were visualized and upto 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extractof ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearinghamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results fromthese in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract ofginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involvedin conferring resistance of CCA to the ginger extract.