Background: Although Leptospira isolation has been reported in Chilean cattle, only serological evidence of serovar Hardjo bovis infection has been routinely reported. The present report provides characterization of the pathological presentation and etiology of a clinical case of leptospirosis in a calf from the Los Rios Region in Chile. Case presentation: In a dairy herd in southern Chile, 11 of 130 calves died after presenting signs such as depression and red-tinged urine. One of these calves, a female of eight months, was necropsied, and all the pathological findings were consistent with Leptospira infection. A urine sample was submitted to conventional bacteriological analysis together with highly specific molecular biology typing tools, in order to unravel the specific Leptospira specie and serovar associated with this clinical case. Conclusion: Leptospira interrogans serovar Hardjoprajitno was associated with the investigated calf clinical case.