Little is known about the role that B cells play in immune responses to infection with the intracellular pathogen, Mycobacterium avium subsp. paratuberculosis (MAP). Traditionally, the role of B cells has been constrained to their function as antibody-producing cells, however, antibodies are not thought to play a protective role in mycobacterial infections. The present study was designed to characterize B cell subpopulations as well as activation/maturation states in cattle with paratuberculosis. Peripheral blood mononuclear cells (PBMCs) were isolated from noninfected control cows (n = 8); as well cattle naturally infected with MAP in the subclinical (n = 8) and clinical (n = 7) stage of infection and stimulated with MAP antigen for 6 days. MAP infection resulted in greater numbers of total B cells for clinical cows compared to control noninfected cows. The major subpopulation in freshly isolated PBMCs in clinical cows was B-1a B cells, but this shifted to a composite of both B-1a and B-2 B cells upon stimulation of PBMCs with either MAP antigen or pokeweed mitogen, with higher numbers of B-2 B cells. Early B cells were observed to predominate the population of B cells in PBMCs, with lesser populations of germinal B cells, memory B cells and plasma cells. These subpopulations were elevated in clinical cows upon stimulation of PBMCs with MAP antigen, except for plasma cells which were lower compared to control noninfected cows. Increased numbers of B cells in clinical cows aligned with higher expression of B cell markers such as MAPK1/3, BTG1, Bcl2, CD79A and SWAP70, depending upon in vitro stimulation with either mitogen or antigen. This would indicate that the B cells were capable of activation but were anti-apoptotic in nature. The shift to B-2 B cells in the periphery of clinical cows seems to be indicative of an expansion of memory B cells, rather than plasma cells. This may be a last attempt by the host to control the rampant inflammatory state associated with advanced clinical disease.