Equine sarcoids are common therapy-resistant skin tumours induced by bovine papillomavirus type 1 or 2 (BPV1, BPV2) infection. We have previously shown that prophylactic vaccination with BPV1 L1 virus-like particles (VLPs) efficiently protects horses from experimental BPV1-induced pseudo-sarcoid development. Here, we assessed BPV1 L1 VLP vaccine-mediated long-term protection from experimental tumour formation in seven horses 5 years after immunization with three different doses of BPV1 L1 VLPs, and three unvaccinated control animals. Horses were challenged by intradermal inoculation with infectious BPV1 virions at 10 sites on the neck (106 virions per injection). In vaccinated horses, BPV1 challenge did not result in any apparent lesions irrespective of vaccine dosage and BPV1-neutralizing antibody titres that had dropped considerably over time and below the detection limit in one individual. Control horses developed pseudo-sarcoids at all inoculation sites. We conclude that immunization of horses with BPV1 L1 VLPs induces long-lasting protection against experimental BPV1 virion-induced disease.