In March 2011, a seroprevalence study of leptospirosis in humans was conducted in Mayotte to obtain reliable population-based estimates of the prevalence of this infection on the island and to identify factors associated with the disease. This was a cross-sectional study using a random cluster sample of the population aged 5 years and older living on the island. Participants were interviewed at their homes, and a blood sample was collected. Laboratory tests (MAT serology) were performed by the National Reference Center (CNR) for Leptospirosis at the Pasteur Institute in Paris. In total, the study involved 729 households, representing 1,420 individuals aged 5 years or older. The seroprevalence of leptospirosis estimated from the study was 16.5%, indicating significant environmental contamination by pathogenic leptospires. Factors associated with the risk of the disease included having lived in Mayotte for more than 10 years, being a farmer or unemployed, being male, having contact with livestock (zebus or goats), and having played in trash bins during childhood. The results of the seroprevalence study confirm that leptospirosis is highly endemic in Mayotte, underscoring the need for surveillance of this disease on the island. (R.A.)