The safety and efficacy of a killed vaccine against bovine paratuberculosis (SILIRUM®, CZ Veterinaria) has been evaluated in calves experimentally infected with Mycobacterium avium subspecies paratuberculosis (Map). Ten calves, out of 18, were injected subcutaneously with a single dose of the vaccine, and the remaining 8 with PBS. Two months after vaccination, 8 and 6 calves from the vaccinated and control groups respectively, were challenged, by oral route, with 6 doses of 6.9 x 10¹⁰ cfu of Map. The remaining calves were kept as controls. Peripheral cellular (IFN-γ production) and humoral immune responses were assessed, as well as fecal Map isolation and PCR identification in blood samples, between 0 and 330 dpv. Three vaccinated and two control calves were slaughtered at 180 dpv (120 dpi) and the remaining 13 at 330 dpv (270 dpi). Pathological and bacteriological studies were conducted in different tissue samples from the intestines and related lymph nodes. Animals were categorised according to the type of lesion present, following previous classifications made in natural bovine paratuberculosis, as focal, multifocal or diffuse. The number of granulomas was counted in sections from the intestine and lymph nodes. No adverse reactions were observed in vaccinated calves. Humoral responses appeared in vaccinated groups at 90 dpv, whereas cellular responses were detected at 30 dpv, reaching the highest values at 120 dpv. At 330 dpv, IFN-γ values were still high. A significant reduction in lesion severity and number of granulomas present in the tissues, was observed in vaccinated calves compared to controls. In the vaccinated, no lesions or lesions confined mainly to the lymphoid tissue were observed, except in one case showing a diffuse form, whereas in control calves, lesions extended to the intestinal mucosa. Isolation of Map from faeces or tissues was reduced in vaccinated animals. The administration of a single dose of SILIRUM® in calves, has demonstrated to be safe and able to control the disease, causing a reduction in the bacterial load and severity and number of lesions.