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Background Leptospirosis is a bacterial zoonosis caused by pathogenic Leptospira for which rats are considered as the main reservoir. Disease incidence is higher in tropical countries, especially in insular ecosystems. Our objectives were to determine the current burden of leptospirosis in Seychelles, a country ranking first worldwide according to historical data, to establish epidemiological links between animal reservoirs and human disease, and to identify drivers of transmission. Methods A total of 223 patients with acute febrile symptoms of unknown origin were enrolled in a 12-months prospective study and tested for leptospirosis through real-time PCR, IgM ELISA and MAT. In addition, 739 rats trapped throughout the main island were investigated for Leptospira renal carriage. All molecularly confirmed positive samples were further genotyped. Results A total of 51 patients fulfilled the biological criteria of acute leptospirosis, corresponding to an annual incidence of 54.6 (95% CI 40.7–71.8) per 100,000 inhabitants. Leptospira carriage in Rattus spp. was overall low (7.7%) but dramatically higher in Rattus norvegicus (52.9%) than in Rattus rattus (4.4%). Leptospira interrogans was the only detected species in both humans and rats, and was represented by three distinct Sequence Types (STs). Two were novel STs identified in two thirds of acute human cases while noteworthily absent from rats. Conclusions This study shows that human leptospirosis still represents a heavy disease burden in Seychelles. Genotype data suggests that rats are actually not the main reservoir for human disease. We highlight a rather limited efficacy of preventive measures so far implemented in Seychelles. This could result from ineffective control measures of excreting animal populations, possibly due to a misidentification of the main contaminating reservoir(s). Altogether, presented data stimulate the exploration of alternative reservoir animal hosts., Author summary Leptospirosis is an emerging environmental infectious disease caused by corkscrew shaped bacteria called Leptospira. Humans usually get infected during recreational or work-related outdoor activities through contact with urine excreted by animal reservoirs. As a zoonotic disease, leptospirosis is a good example of the One Health concept for it links humans, animals and ecosystems in a web of pathogen maintenance and transmission. This zoonosis is highly prevalent in the tropics and especially in tropical islands. Seychelles archipelago has been reported as the country with highest human incidence worldwide, although figures are based on dated studies and/or poorly specific tests. The presented investigation aimed at providing an updated information on human leptospirosis burden in Seychelles and exploring the transmission chains in their environmental aspects. Presented data confirms that the disease still heavily impacts the country. Genotyping of pathogenic Leptospira in human acute cases reveals that three distinct Sequence Types (STs) are involved in the disease. However, rats typically considered as the main reservoir in Seychelles, harbor only one of these STs, found only in a minority of human cases. Hence, it appears that rats are likely not the main reservoir of leptospirosis in Seychelles, which has important consequences in terms of preventive measures to be implemented for a better control of human leptospirosis.