Infectious disease during transition and early lactation periods has the potential to decrease production performance and increase morbidity and mortality in dairy cows. Enhanced adaptive immunity through nutritional supplementation may counter immunosuppression that occurs naturally during the periparturient and early lactation periods. The objective of this study was to evaluate the effect of a direct-fed microbial, Bacillus pumilus 8G-134, on systemic immunity in dairy cows through flow cytometric analysis of peripheral blood mononuclear cells. Thirty cows were fed a control diet or a diet including 5 × 10⁹ cfu/head/day B. pumilus 8G-134 from 3 weeks prepartum to 22 weeks postpartum. Peripheral blood mononuclear cells were collected at 60 d in milk and analyzed by flow cytometry. Leukocytes and lymphocytes were gated based on forward and side scatter, and relevant cell surface markers were examined in each population. B. pumilus 8G-134 supplementation increased (P < 0.05) expression of T cell surface markers CD4, CD8, CD62L, CD25, and CD45RO, but did not affect proportions of T cell subsets. There was no effect on expression of cell surface markers on innate immune cells (Mono, CD14, CD45, CD172a, and CD62L) or B cells (Bcell, and CD21). Feeding B. pumilus 8G-134 induced expression of T cell surface markers that are naturally suppressed during parturition. By targeting the adaptive immune system, B. pumilus 8G-134 was able to enhance T cell marker expression without activating a systemic immune response that would adversely affect energy balance. This may help dairy cows recover faster from periparturient immunosuppression.