Mechanism of Mycobacterium avium subsp. paratuberculosis (Map) invasion through intestinal mucosa is not completely understood. In the present study, we developed an in vivo multiple-intestinal loop model in lambs to investigate (i) the type of cells involved in the bacterial uptake across the intestinal mucosa, (ii) the efficiency of bacterial uptake in different segments of the small intestine and (iii) the ability of different strains of Map to invade the various segments of the small intestine. Four loops on ileum and four loops each on Peyer's patch and non-Peyer's patch areas of jejunum were constructed by surgical procedure. The caprine, bovine, and vaccine strains of Map were used for infection. Map-infected intestinal loop tissues were collected at 1, 3, 6, 12, and 24 h post-infection and processed for electron microscopy, histology, bacterial culture and bacterial counting. All these parameters revealed that Map invaded through M cells and the enterocytes and bacterial translocation across M cells was greater than the enterocytes. Bacterial invasion was greater in ileal loops when compared to jejunal loops. Within the jejunal loops, bacterial uptake was higher in Peyer's patch areas than that of non-Peyer's patch areas. The caprine and bovine strains of Map showed greater ability for invasion into the small intestinal mucosa than that of the vaccine strain.