A cross-sectional study was conducted in Mapastepec, a rural community of the southern state of Chiapas, Mexico. The overall prevalence of leptospirosis infection in 1169 subjects was 37.7% [95% confidence intervals (95% CI) 34.9-40.5]. The main risk factors related to leptospirosis infection were flooding, mainly if subjects had a skin cut or abrasion [odds ratio (OR) 4.2; 95% CI 3.1-5.7], having domestic animals, either dogs and/or cats (OR 1.3; 95% CI 0.96-1.8) or cattle and/or pigs (OR 1.9; 95% CI 1.3-2.7), contact with animal excreta with no protection and with a skin cut or abrasion (OR 2.3; 95% CI 1.1-4.6). Those subjects with a dengue infection in the previous year had also an excess risk (OR 1.4; 95% CI 0.9-2.0). Mapastepec is a previously unknown area with high endemicity. Specific preventive measures should be adopted to prevent any contact with infected animals, and animal immunization should also be implemented. There is need of an epidemiological surveillance system to allow proper diagnosis.