Liver fluke infection of Opisthorchis viverrini (O. viverrini) is closely associated with several hepatobiliarydiseases including cholangiocarcinoma (CCA), but no reports have described these diseases in chronic andlong-standing experimental opisthorchiasis in hamsters more than 10 months of age. A longer period of infectioncould induce different pathological lesions. To prove the hypothesis, we therefore sequentially investigatedhistological changes of the hepatobiliary system in 4 groups of hamster: O. viverrini infection (OV group) for upto 20 months, O. viverrini infection combined with short-term DMN (OV+DMN group) until 7 months, longtermtreatment with DMN (DMN group) to 7 months and normal control group for up to 20 months. Pathologicalchanges in hamsters of the OV group gradually increased. Induction of CCA in this study was apparent with allthree protocols. Importantly, this is the first report of CCA-induction in hamsters solely with long-termopisthorchiasis for up to 20 months. Although the histopathology of CCA in the OV group showed some differencesin appearance from the OV+DMN and DMN groups, overall, O. viverrini itself can really induce CCA. Inaddition, this study confirms the previous studies both in vitro and in vivo on of effects of parasites and theirmetabolic products inducing cell proliferation, resulting in cholangiocarcinogenesis.