Leptospirosis is a worldwide zoonotic disease, but feline leptospirosis is rarely reported. This study aimed at investigating Leptospira spp. prevalence in cats from southern Italy, evaluating risk factors, clinical findings and laboratory data associated with infection. The serum of 112 cats was investigated by microscopic agglutination test (MAT), detecting anti-Leptospira antibodies against 14 pathogenic serovars. Blood and urine samples were tested by a real-time polymerase chain reaction targeting the lipL32 gene of pathogenic Leptospira. Antibodies against serovars Poi, Bratislava, Arborea, Ballum, Pomona and Lora were detected in 15.3% (17/111) of cats (titers range: 20-320). Leptospira spp. DNA was found in 3% (4/109) of blood and 9% (10/111) of urine samples. The spring season was the only risk factor for urinary Leptospira DNA shedding. Laboratory abnormalities significantly associated and/or correlated with Leptospira spp. positivity were anemia, monocytosis, neutrophilia, eosinopenia, increased alanine aminotransferase activity, hypoalbuminemia and hyperglobulinemia. In the investigated areas, cats are frequently infected by Leptospira spp. and can represent an additional reservoir or sentinel for a risk of infection. Moreover, some laboratory changes could be compatible with a pathogenic effect of Leptospira spp. in the feline host.