question: Exposure of neonates to Mycobacterium avium subsp. paratuberculosis (MAP) via infected dams is the primary mode of transmission of Johne’s disease. Little is known about the impacts of feeding colostrum and supplemental vitamins on the gut microbiome in calves exposed to MAP. In the present study, calves were assigned at birth to one of six treatment groups: (1) Colostrum deprived (CD), no vitamins; (2) colostrum replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D(3); (5) CR, vitamin E; (6) CR, vitamins A, D(3), E, with five calves per treatment in a 14-day study. All calves were orally inoculated with MAP on days 1 and 3 of the study. Differences due to vitamin supplementation were not significant but treatment groups CR-A, CR-E, and CR-ADE had higher numbers of MAP-positive tissues overall. Shannon diversity indices demonstrated regional differences in microbial communities, primarily Proteobacteria, Bacteroidetes, and Firmicutes, between the ileum, cecum, and spiral colon of all calves. CD calves exhibited increased richness compared with CR calves in the cecum and spiral colon and harbored increased Proteobacteria and decreased Bacteroidetes in the mucosa compared with the lumen for all three tissues. Overall, supplementation with vitamins did not appear to influence gut microbiome or impact MAP infection. Feeding of colostrum influenced gut microbiome and resulted in fewer incidences of dysbiosis.