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Background: Porteirinha is endemic for visceral leishmaniasis (VL), with intense disease transmission of the disease. We evaluated the impact of canine euthanasia as a single control measure on the incidence of VL in humans and canines. Methods: A prospective observational cohort study was carried out over four years (1998–2002) in 8 of the 12 neighborhoods of the city. The dynamics of canine visceral leishmaniasis (CVL) transmission were evaluated for 2 years, before beginning the screening–culling intervention. The comparative morbidity index (CMI) was used to stratify areas with the greatest risk of CVL, and the spatial distribution of human and canine VL cases was compared using univariate and bivariate K-functions. Results: Human cases conglomerated in three neighborhoods. Spatial clusters were detected for CVL in 1998, 2000, and 2001, but not in 1999, when greater spatial dispersion occurred. The screening and culling intervention reduced the number of human VL cases and decreased the incidence of CVL, mainly in neighborhoods with a high CMI. Conclusions: The systematic euthanasia of seropositive dogs was shown to be an effective control action of the Program for Control of Visceral Leishmaniasis (PCLV) in Brazil. The fundamental role of domestic dogs in the epidemiological chain of VL was reaffirmed.