Zoonotic leptospirosis is an infectious disease caused by pathogenic Leptospira bacteria that can be transmitted from animals to humans. Animals, especially wild and domestic mammals, are reservoir hosts that carry and excrete the bacteria in their urine. A retrospective analysis of the incidence of leptospirosis in animals and humans in Ukraine from 2003 to 2022 was conducted based on reports from regional state laboratories of veterinary medicine and the Center for Public Health. GIS mapping was used to analyze and visualize the data. The incidence and prevalence of leptospirosis among different animal species, including farm, domestic and wild animals, were analyzed. These studies included a retrospective analysis of blood serum samples from different animal species for leptospirosis and analysis of the results to determine the level of seropositivity. The study analyzed a total of 6,543,934 samples, with an overall seropositivity rate of 3.3%. Among cattle, 4.1% of the samples were positive. For pigs, 2.1% were positive. Horses had a seropositivity rate of 7.9%, while dogs and cats had seropositivity rates of 17.5% and 13.1% respectively. Wild boars showed a positivity rate of 2.2%. The number of seropositive animals of different species in Ukraine showed different dynamics from 2003 to 2022. The highest number of positive cases was found among cattle, pigs and horses. However, the number of positive cases decreased significantly after 2015, which coincides with a significant reduction in the number of farm animals. Trends of increasing seropositivity among dogs and cats in the period 2007–2015 indicate that these species are also active carriers of Leptospira, as are the leading agricultural species. Wildlife species with stable seropositivity serve as both sources and reservoirs of Leptospira. Wild pigs are becoming the main bacterial carriers in all regions of Ukraine. There is a marked disproportion between the number of positive cases among farm animals and wildlife, indicating that seropositivity in these populations exists largely autonomously. A total of 7,937 people were infected in Ukraine between 2003 and 2022. Certain regions, such as Lviv, Kherson, Kyiv, Mykolaiv, and Vinnytsia, account for a larger share of the total number of cases, ranging from 4.8% to 7.3%. The incidence of human leptospirosis has fluctuated over the years, peaking at 473 cases in 2014, followed by a decline: 301 cases in 2015, 323 cases in 2016 and 330 cases in 2017, and in subsequent years the number of cases remained relatively stable, ranging from 142 to 295 cases per year. The study emphasizes the need to improve preventive measures and raise public awareness of the disease, especially in rural areas. It is important to adopt a comprehensive interdisciplinary approach involving collaboration between veterinary medicine, public health and environmental authorities to better understand and address the complexities of leptospirosis transmission and reduce its impact on animal and human health.