A role of wildlife species as paratuberculosis reservoirs is strongly suspected based on field and molecular epidemiologic evidence. This paper presents the first large-scale data on enzyme-linked immunosorbent assay (ELISA) against Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in red deer from Spain, and tests the effect of host and environmental risk factors on antibody levels. A total of 257 out of 852 serum samples tested positive, yielding a total seroprevalence of 30.16% (95% CI 27.08-33.24). Sampling locality, presence of cattle and increasing age explained the variation in the individual ELISA optical density (OD) results. Data presented in this study strongly suggest that Spanish red deer are exposed to MAP. While contact with cattle was statistically significant, some wild populations showed the highest positivity to the ELISA. The results support the need of a careful study of MAP prevalence based on culture and molecular tools in order to clarify if deer play a significant role as paratuberculosis reservoirs for livestock, and if deer paratuberculosis is affecting hunting harvest, trophy quality, or wild animal welfare in Spain.