Immune system development during gestation and suckling is significantly modulated by maternal environmental and dietary factors. Breastfeeding is widely recognized as the optimal source of nutrition for infant growth and immune maturation, and its composition can be modulated by the maternal diet. In the present work, we investigated whether oral supplementation with Bifidobacterium breve M-16V and short-chain galacto-oligosaccharide (scGOS) and long-chain fructo-oligosaccharide (lcFOS) to rat dams during gestation and lactation has an impact on the immune system and microbiota composition of the offspring at day 21 of life. On that day, blood, adipose tissue, small intestine (SI), mesenteric lymph nodes (MLN), salivary gland (SG), cecum, and spleen were collected. Synbiotic supplementation did not affect the overall body or organ growth of the pups. The gene expression of Tlr9, Muc2, IgA, and Blimp1 were upregulated in the SI, and the increase in IgA gene expression was further confirmed at the protein level in the gut wash. Synbiotic supplementation also positively impacted the microbiota composition in both the small and large intestines, resulting in higher proportions of Bifidobacterium genus, among others. In addition, there was an increase in butanoic, isobutanoic, and acetic acid concentrations in the cecum but a reduction in the small intestine. At the systemic level, synbiotic supplementation resulted in higher levels of immunoglobulin IgG2c in plasma, SG, and MLN, but it did not modify the main lymphocyte subsets in the spleen and MLN. Overall, synbiotic maternal supplementation is able to positively influence the immune system development and microbiota of the suckling offspring, particularly at the gastrointestinal level.