Lyme borreliosis (LB) is a multi-systemic tick-borne disease affecting both humans and animals, including horses, and is caused by a group of interrelated spirochetes classified within the Borrelia burgdorferi sensu lato (s.l.) complex. Despite the high reported seroprevalence in the European equine population for B. burgdorferi s.l., to-date no documented clinical cases have been described. A 6-year-old Paint gelding was referred with a history of three weeks of fever, intermittent lameness and digital flexor tendon sheath effusion of the right hind limb. Based on a strict diagnostic protocol, which included serological tests for infectious diseases and molecular investigations, a final diagnosis was made of polysynovitis due to B. burgdorferi s.l. infection. An unreported aspect observed in this case was the absence of the pathogen DNA in two of the affected joints. To the authors’ knowledge, the case described represents the first documented clinical case of equine LB in Italy. Moreover, the absence of pathogen DNA in two of the affected joints observed in this case revealed a possible similarity with the same condition described in humans, where an immunomediated pathogenesis for arthropathy due to B. burgdorferi s.l. infection is suspected. Since humans and horses share the same habitat, this report supports the role of the horse as potential sentinel for human biological risk.