Cholangiocarcinoma (CCA) is an aggressive， metastatic bile duct cancer. CCA is difficult to diagnose， and responds poorly to current radio- and chemo-therapy. Piperlongumine (PL) is a naturally-occurring small molecule selectively toxic to cancer cells by targeting reactive oxygen species (ROS). In this study， we demonstrated the potential anticancer activity of PL in CCA. PL markedly induced death in CCA cell lines in a dose- and time-dependent manner through the activation of caspase-3 and PARP. PL also stimulated ROS accumulation in CCA. Co-exposure of PL with the ROS scavenger N-acetyl-L-cysteine or GSH completely blocked PL-induced apoptosis in CCA cell lines. Increased p21 via the p53-independent pathway in PL-treated CCA cells led to G2/M phase arrest and cell apoptosis. In addition， the study showed that PL trigger CCA cell lines death through JNK-ERK activation. Furthermore， the different antioxidant capacity of CCA cell lines also indicates the susceptibility of the cells to PL treatment. Our findings reveal that PL exhibits anti-tumor activity and has potential to be used as a chemotherapeutic agent against CCA.