Abstract Objectives To evaluate the cytotoxic activity of atractylodin and its potential effects on heme oxygenase (HO)-1 production, STAT1/3 phosporylation and major NF-κB protein expression in the cholangiocarcinoma-associated cell line CL-6. Methods Standard MTT assay was used for accessing antiproliferative activity on CL-6 cells. Normal human embryonic fibroblast (OUMS) cell was taken as control cell line. Colony formation and wound healing assay were conducted to access the effects of atractylodin on cell proliferation and directional migration activity of CL-6 cells. Western blot was used for evaluating levels of protein expression and phosphorylation. Key findings Atractylodin exhibited selective cytotoxicity towards CL-6 as compared with OUMS with IC50 of 216.8 (212.4-233.8) and 351.2 (345.7-359.5) μm [median (range)], respectively. Exposure to the compound dose-dependently inhibited colony formation ability and decreased wound closure potential of CL-6 cells. Atractylodin treatment suppressed HO-1 production in CL-6 cells. It dose-dependently inhibited STAT1/3 protein phosphorylation and moderately inhibited NF-κB (p50), NF-κB (p52), and NF-κB (p65) protein expression in both dose- and time-dependent manner. Conclusions Atractylodin exerts significant cytotoxic activity against CL-6 cells which may be linked to its suppressive effect on HO-1 production, STAT1/3 phosphorylation and expression of key NF-κB proteins.