There has been a strong, positive correlation between opisthorchiasis-associated cholangiocarcinoma andinfection with Helicobacter. Here a rodent model of human infection with Opisthorchis viverrini was utilized tofurther investigate relationships of apparent co-infections with O. viverrini and H. pylori. A total of 150 hamsterswere assigned to five groups: i) Control hamsters not infected with O. viverrini; ii) O. viverrini-infected hamsters;iii) non-O. viverrini infected hamsters treated with antibiotics (ABx); iv) O. viverrini-infected hamsters treatedwith ABx; and v) O. viverrini-infected hamsters treated both with ABx and praziquantel (PZQ). Stomach,gallbladder, liver, colonic tissue, colorectal feces and O. viverrini worms were collected and the presence ofspecies of Helicobacter determined by PCR-based approaches. In addition, O. viverrini worms were cultured invitro with and without ABx for four weeks, after which the presence of Helicobacter spp. was determined. In situlocalization of H. pylori and Helicobacter-like species was performed using a combination of histochemistry andimmunohistochemistry. The prevalence of H. pylori infection in O. viverrini-infected hamsters was significantlyhigher than that of O. viverrini-uninfected hamsters (p≤0.001). Interestingly, O. viverrini-infected hamsters treatedwith ABx and PZQ (to remove the flukes) had a significantly lower frequency of H. pylori than either O. viverriniinfectedhamsters treated only with ABx or O. viverrini-infected hamsters, respectively (p≤0.001). QuantitativeRT-PCR strongly confirmed the correlation between intensity H. pylori infection and the presence of liver flukeinfection. In vitro, H. pylori could be detected in the O. viverrini worms cultured with ABx over four weeks. Insitu localization revealed H. pylori and other Helicobacter-like bacteria in worm gut. The findings indicate thatthe liver fluke O. viverrini in the biliary tree of the hamsters harbors H. pylori and Helicobacter-like bacteria.Accordingly, the association between O. viverrini and H. pylori may be an obligatory mutualism.