STAT3 plays a significant role in the development of cholangiocarcinoma (CCA) associated with the liver fluke (Opisthorchis viverrini; Ov). Xanthohumol (XN), a prenylated flavonoid extracted from hops, has known anticancer activity and could potentially target STAT3. The present study determined the effect of XN on STAT3, as well as ascertained its usefulness against CCA. The CCA cell proliferation at 20 M and 50 M of XN was shown to inhibited, while 20 M partially inhibited IL-6-induced STAT3 activation. At 50 M, the inhibition was complete. The reduction in STAT3 activity at 20 and 50 M was associated with a significant reduction of CCA cell growth and apoptosis. We also found that the administration of 50 M XN orally in drinking water to nude mice inoculated with CCA led to a reduction in tumor growth in comparison with controls. In addition, apoptosis of cancer cells increased although there was no visible toxicity. The present study shows that XN can inhibit STAT3 activation both in vivo and in vitro due to suppression of the Akt-NFB signaling pathway. XN should be considered as a possible therapeutic agent against CCA.