Cholangiocarcinoma (CCA) is a progressively fatal form of cancer originating from the malignant transformation of hepatic biliary cholangiocytes. The present study reports for the first time in vitro growth inhibitory activities of β‐eudesmol, the bioactive sesquiterpenoid present in the rhizome of Atractylodes lancea (Thunb) DC., with respect to its underlying potential effects on heme oxygenase‐1 (HO‐1) production, STAT1/3 phosphorylation, and NF‐κB protein expression in human CCA cell line CL‐6. The cytotoxic effect of β‐eudesmol on CL‐6 cells was evaluated by MTT assay using normal human embryonic fibroblast (OUMS) as a control cell line. Results indicated that β‐eudesmol exhibited selective cytotoxicity towards CL‐6 compared to OUMS with mean (±SD) IC50 (concentration that inhibits cell growth by 50%) values of 166.75 ± 3.69 and 240.01 ± 16.54 μmol/L, respectively. In addition, it also significantly suppressed colony forming and wound healing ability of CL‐6 cells in a concentration‐dependent manner. Western blot analysis indicated that β‐eudesmol treatment resulted in significant suppression of HO‐1 production in CL‐6 cells. Its inhibitory effects on the phosphorylation of STAT1/3 proteins and expression of NF‐κB (p65 and p50) proteins were concentration‐dependent. Taken together, these results suggest that β‐eudesmol exerts significant growth inhibitory activity on CL‐6 cells that may be linked to its inhibitory effect on the production of HO‐1, phosphorylation of STAT1/3, and expression of major NF‐κB proteins.