Leptospirosis has been reported in rural areas of Brazil. However, there is limited information about the exposure risk or the risk of Leptospira infection for rural-based populations. A cross-sectional study was carried out in order to determine the prevalence and risk factors for prior Leptospira infection in a rural subsistence farming region of the state of Rio Grande do Norte, an area in which outbreaks of leptospirosis have occurred. Among 290 individuals enrolled, 44 (15.2%) had anti-Leptospira IgM antibodies as determined by IgM ELISA. Infection tended to occur with activities related to the rice fields (P = 0.08). Our findings indicate that Leptospira infection occurs even in years of low rainfall, and may have an important impact among poor rural-based subsistence farmers in Brazil. Additional studies are needed to characterize the mode of transmission in this region.