Galacto‐oligosaccharides (GOS) are prebiotics that support the growth of bifidobacteria and lactobacilli. During influenza vaccination, mice supplemented with GOS had increased T helper (Th) 1 delayed‐type hypersensitivity biomarkers. The objective of our study was to determine the effects of GOS on gastrointestinal and systemic immune cell populations. We hypothesized that GOS would support Th1 responses by increasing CD8+ T cell and Natural Killer (NK) cell numbers. We gavaged 129/Sv SMAD3 knockout mice with 5,000mg GOS/Kg body weight/day for 7 and 14 days and excised blood, spleen, mesenteric lymph nodes (mLN), and Peyer's patches (PPs). Lymphocytes were isolated and stained for flow cytometric analysis of Th cells (CD3+/CD4+), cytotoxic T cells (CD3+/CD8+), B cells (CD19+), and immature (NKp46+/DX5−) and mature NK cells (NKp46+/DX5+). GOS treatment increased total CD4+ T cells in the PPs and mLN within 1 week. GOS also increased CD8+ cytotoxic T lymphocytes in PPs by 1 week and blood by 2 weeks. Mature NK cells were increased in the blood after 2 weeks of GOS treatment. Therefore, GOS treatment modulates both gastrointestinal and systemic immune cell populations. The immune response is Th1 biased during steady‐state conditions in SMAD3 knockout mice treated with GOS. Therefore, GOS may be useful prophylactics and therapeutics. This study was generously supported by GTC Nutritionals.