Background: Liver fluke infection caused by the parasite Opisthorchis viverrini (O. viverrini), a humancarcinogen, is endemic in north-eastern Thailand and remains a major health problem. Objectives: The objectivesof the study were to (1) resurvey the prevalence of O. viverrini infection in a field site from the Khon Kaen CohortStudy (in newly recruited subjects as well as previous cohort subjects surveyed in 1992); (2) investigate howsubjects’ lifestyle habits and their exposure to health promotion initiatives influence changes in prevalence ofO. viverrini infection. Materials and Methods: The prevalence of O. viverrini infection in the cohort subjects (aswell as new subjects) was investigated using faecal egg counts. Information on demographic factors, lifestyle andawareness of health promotion initiatives were obtained through questionnaires. Results: O. viverrini infectionrates in the same individuals of the cohort were lower in 2006 than in 1992. Also, by studying the period effect, thecurrent 35-44 year olds had a 12.4% (95% CI 3.9% to 20.9%) lower prevalence of O. viverrini infection than the35-44 year olds in 1992 (24.2% versus 11.8%). Lifestyle choices showed that smoking and alcohol consumptionwere associated with an increased chance of acquiring O. viverrini infection with adjusted odds ratios of 10.1(95%CI 2.4-41.6) and 5.3 (95%CI 1.2-23.0), respectively. Conclusions: Our study has demonstrated that althoughthe prevalence of O. viverrini infection over a 14-year period has decreased, unhealthy lifestyle was commonwith smoking and alcohol consumption being associated with increased chances of infection, emphasising thedouble burden of disease which developing countries are facing.