A cross-sectional seroprevalence study on leptospirosis, using microscopic agglutination test (MAT), was conducted in rural villages in Khammouane Province, Lao People’s Democratic Republic, in December 2006. The overall prevalence of leptospiral infection among 406 subjects was 23.9% (95% confidence interval [CI] 19.7–28.0%). Independent risk factors for the infection, identified by multivariate logistic regression, were male sex (odds ratio [OR], 1.92; 95% CI: 1.24–2.98), recent flooding on one’s own property (OR, 2.12; 95% CI: 1.25–3.58), and collecting wood in the forest (OR, 1.90; 95% CI: 1.17–3.09). Age, occupation, and animal ownership were not associated with seropositivity. Flooding was associated with the risk of infection particularly for women, whose behaviors or activities involving contact with floodwater were presumed to play an important role. This study showed that leptospirosis is endemic in Khammouane Province and that local flooding plays an important role in the transmission of the disease.