Equine sarcoids are therapy-resistant neoplasms in equids that are induced by bovine papillomavirus types 1 or 2 (BPV1, BPV2). They are locally invasive, fibroblastic tumours of the dermis and can occur anywhere on the body of horses, zebras, donkeys and mules. Up to 12% of tumours diagnosed in horses are sarcoids, so the need of a vaccine is great mainly because possible treatments are not consistently effective. Recently we have shown that immunization of horses with BPV1 L1 virus-like particles is highly immunogenic, safe and protects the animals from pseudo-sarcoid development induced by experimental BPV1 infection. In the herein presented study we assessed BPV1 L1 VLP-mediated long-term protection from experimental tumour formation. Five years after third immunization with BPV1 L1 VLPs (50, 100 or 150 μg in alum) we challenged seven immunized horses and three naive control horses with wild-type BPV1 virions at ten sites of the neck (106 virions per injection). Blood was taken before inoculation and we determined that antibody titres had dropped over time and even below detection limit in one horse. Despite this finding BPV1 challenge had no apparent effect in vaccinated horses regardless of antibody titres and VLP dosage, while all control horses developed pseudo-sarcoids at all inoculation sites. Together with previous findings on memory T-cell activation in non-lymphoid tissues we propose that long-term protection of immunized horses is mainly based on immune cell memory conferred by the BPV1 L1 VLP-based vaccine.