A 12-month study was conducted to evaluate the effects of feeding a low calcium (Ca) diet or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) infusion on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Male beige mice 6–8 weeks of age were assigned to one of the following treatments: (1) non-infected, (2) infected, (3) non-infected/1,25(OH)2D3, (4) infected/1,25(OH)2D3, and (5) infected/low Ca (0.15%) diet. Infected mice were inoculated intravenously with live M. paratuberculosis. At 1, 6 and 12 months postinfection, mice in Treatments 3 and 4 were implanted subcutaneously with mini-osmotic pumps to deliver 1,25(OH)2D3. Infusion with 1,25(OH)2D3 exacerbated M. paratuberculosis infection in most tissues at all time points. Mice infused with 1,25(OH)2D3 had higher bacterial counts in spleen, liver, and ileum compared with control infected mice after 1 month of infection. In contrast, feeding a low Ca diet reduced the number of viable organisms cultured from the liver and ileum of infected mice. Plasma Ca and 1,25(OH)2D3 were increased in mice infused with 1,25(OH)2D3 at all time points but values for low Ca mice were not different than for non-infused mice. Splenocyte production of TNF, IL-1 and IL-6 was higher for mice fed the low Ca diet compared with control infected mice after 1 month of infection. Inducible IL-6 activity remained higher for this treatment at 6 months postinfection. These results suggest that feeding a low Ca diet to mice chronically infected with M. paratuberculosis appears to enhance their ability to clear the infection in a manner distinct from any effect of 1,25(OH)2D3.