Previous studies demonstrated that peanut testa extracts (KK4 and ICG15042) containing natural histone deacetylase (HDAC) inhibitors inhibited the growth of several human cancer cell lines via apoptosis induction. The aims of this study were to investigate the anti-proliferative effects and the mechanism(s) responsible for apoptosis induction mediated by these peanut testa extracts in human cholangiocarcinoma cell lines (KKU-M214 and KKU-100). The anti-proliferative effects were assessed by MTT assay. Apoptotic cell death and cell cycle arrest were analyzed by flow cytometry. The caspase activities were studied using colorimetric caspase activity assay and western blot analysis. Our results revealed that KK4 and ICG15042 extracts inhibited cell proliferation of both KKU-M214 and KKU-100 cells in a dose- and time-dependent manner, with IC₅₀ values of 38.28 ± 0.29 (KK4), 43.91 ± 1.94 (ICG15042) μg/mL for KKU-M214 and 78.40 ± 1.74 (KK4), 82.77 ± 0.94 (ICG15042) μg/mL for KKU-100 at 72 h. Apoptosis induction by these peanut testa extracts were observed in both KKU-M214 and KKU-100 cells in a concentration-dependent manner. Moreover, the percentage of cells in the sub-G1 phase was significantly increased in both KKU-M214 and KKU-100 cells. Cell cycle arrest was not observed in other cell cycle phases. Activation of caspases 8 and 3 were apparent integral parts of apoptosis induction in both cells. Both peanut testa extracts also caused down-regulation of p53, p21, Bcl-2 and pERK1/2 protein expression in these cells. These results suggest that peanut testa extracts may be potential anti-cancer agents for cholangiocarcinoma chemoprevention or chemotherapy.