Leptospirosis is a reemerging zoonosis of global distribution and is one of the causes of hemorrhagic fevers in the tropics. We sought to determine seroprevalence in humans and animals and isolate Leptospira interrogans sensu lato in domestic animals, rodents, and water sources. The study was conducted in a tropical area of the middle Sinú in Cordoba, Colombia. In a prospective descriptive study, we collected blood and urine from pigs and dogs, sera from rural human workers, sera and kidney macerates of rodents, and water samples from environmental sources. We used microagglutination to screen for antibodies to 13 serovars. Strains were cultured on the Ellinghausen-McCullough-Johnson-Harris medium and confirmed by PCR amplifying lipL32 gene. Seroprevalence was 55.9% in pigs, 35.2% in dogs, and 75.8% in humans; no antibody was detected, and no Leptospira were isolated from kidney macerates of rodents. Seven L. interrogans sensu lato strains were isolated: three from pigs, two from dogs, and two from water. High seroprevalence in pigs, dogs, and humans, concomitant to isolation of strains, demonstrates that in Cordoba, transmission exists among animals, the environment, and humans, which warrants the implementation of public health intervention measures to reduce the epidemiological impact of leptospirosis in the region.