Background Leptospirosis is a widespread but underreported cause of morbidity and mortality. It has rarely been reported in either humans or animals in Madagascar. Methods We conducted a cross-sectional survey of the inhabitants in Moramanga, Madagascar, in June 2011, to estimate the prevalence of human infection using the microscopic agglutination test (MAT). This activity was carried out as part of a workshop implemented by the Pasteur Institute of Madagascar, focusing on surveillance with a one week field study and targeting the health staff of the district level. Results In total, we sampled 678 inhabitants from 263 households. The sex ratio (M/F) was 0.65 and the mean age 26.7 years. We obtained a value of 2.9% for the first recorded seroprevalence of this disease in the human community of Moramanga. Questionnaire responses revealed frequent contacts between humans and rodents in Moramanga. However, activities involving cattle were identified as a risk factor significantly associated with seropositivity (OR=3). Conclusion Leptospirosis remains a neglected disease in Madagascar. This study highlights the need to quantify the public health impact of this neglected disease in a more large scale, in all the country and to establish point-of-care laboratories in remote areas.