Cholangiocarcinoma (CCA) is an uncommon adenocarcinoma which arises from the epithelial cells of the bile ducts. The aim of the study was to investigate the cytotoxicity, toxicity, and anticancer activity of a crude ethanolic 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 the DPPH assay. Investigation of apoptotic activity was performed by DNA fragmentation assay and induction of genes 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 body weight daily or every alternate day for 30 days. Control groups consisting of 10 hamsters for each group were fed with 5-fluorouracil (positive control) or distilled water (untreated control). Median IC50 (concentration that inhibits cell growth by 50%) values for cytotoxicity and anti-oxidant activities of the crude ethanolic extract of ginger were 10.95, 53.15, and 27.86 μg/ml, respectively. More than ten DNA fragments were visualized and up to 7-9 fold up-regulation of MDR1 and MRP3 genes was observed following exposure to the ethanolic extract of ginger. Acute and subacute toxicity tests indicated absence of any significant toxicity at the maximum dose of 5,000 mg/kg body weight given by intragastric gavage. The survival time and survival rate of the CCA-bearing hamsters were significantly prolonged compared to the control group (median of 54 vs 17 weeks). Results from these in vitro and in vivo studies thus indicate promising anticancer activity of the crude ethanolic extract of ginger against CCA with the absence of any significant toxicity. Moreover, MDR1 and MRP3 may be involved in conferring resistance of CCA to the ginger extract.