American visceral leishmaniasis (VL) is a vector-borne disease caused by Leishmania infantum (syn. chagasi) and transmitted by Lutzomyia longipalpis and Lutzomyia evansi phlebotomine sand flies. Dogs not only are the main host reservoirs of the parasite but also suffer the disease; therefore, canine VL (CVL) has assumed an important role in public health. In Colombia, human and CVL are restricted to two transmission foci: one in the north region (Caribbean coast) and other in the central south region (middle Magdalena River Valley). We present a CVL case involving a 2-year-old male dog with a history of lack of appetite, general weakness, and progressive loss of weight. A diagnosis of CVL was obtained using the direct parasitological examination in spleen and bone marrow samples stained with Giemsa and RT-qPCR. The infecting Leishmania species was identified as L. infantum by PCR-restriction fragment length polymorphism amplifying the Hsp70 gene from bone marrow and spleen samples and confirming by sequencing. The patient responded favorably to treatment with intramuscular meglumine antimoniate at a dose of 100 mg/kg daily for 8 weeks and oral allopurinol at a dose of 10 mg/kg every 12 hours until new indication. This is the first report of urban CVL in the city of Cali, Colombia, highlighting the need for surveillance and control programs in the municipalities of the department of Valle del Cauca, a region where VL has not been informed before. The findings also indicate the need to reinforce the surveillance programs in other rural and urban regions of the country where favorable eco-epidemiological conditions exist.