Background: Cholangiocarcinoma is one of the most difficult malignancies for cure. An important prognostic factor for cholangiocarcinoma is metastasis, which precludes curative surgical resection. The development of metastasis requires the movement and invasion of cancer cells from the primary tumor into the surrounding tissue. Recent evidence indicates that capsaicin has depression effects on cancer cell migration and invasion. Thus, we investigated the molecular mechanism of capsaicin-induced anti-migration and anti-invasion effects in HuCCT1 cells. Methods: The invasion, migration and gelatin zymography assay were performed to identify the effect of capsaicin on HuCCT1 cells. We further employed immunoprecipitation, immunoblot and RT-qPCR analysis to study its molecular mechanisms of action. Results: The treatment of capsaicin significantly suppressed tumor migration and invasion. We further found that capsaicin reduced the PMA-induced expression of MMP-9 and MMP-2 but did not alter TIMP-1 and TIMP-2 levels in mRNA and protein. Interestingly, capsaicin elevated the accumulation of NAD+ by AMPK activation in intracellular and enhanced SIRT1 protein levels in nucleus. In addition, capsaicin inhibited NF-κB activity by the deacetylation of the p65 subunit of NF-κ B through SIRT1 activation. Conclusion: These results suggest that capsaicin suppressed PMA-induced up-regulation of MMP9 by inhibition of the MMP9 transcription factor NF-kB through AMPK-NAD+-SIRT1 signaling cascade, and contributes to cholangiocarcinoma cell metastasis.