The nasal mucosa is the body's first barrier against pathogens entering through the respiratory tract. The respiratory immune system of pigs has more similarities with humans than the mouse respiratory system does, and so was selected as the animal model in the present study. To evaluate the effects of Bacillus subtilis as a potential probiotic to stimulate local immune responses, piglets were intranasally administered with Dylight 488-labeled B. subtilis (WB800-green fluorescent protein). The results revealed that B. subtilis was able to reach the lamina propria of the nasal mucosa, nasopharyngeal tonsils and soft palate tonsils. Piglets were subsequently administered intranasally with B. subtilis (WB800) at 3, 12 and 28 days. The results revealed that, following administration with B. subtilis, the number of dendritic cells, immunoglobulin A+ B cells and T cells in the nasal mucosa and tonsils significantly increased (P<0.05). No obvious differences were observed in the morphological structure following B. subtilis administration. There were no statistical differences were observed in the expression of interleukin (IL)-1β, tumor necrosis factor-α and IL-8 mRNA between the B. subtilis treated group and the control group in the nasal mucosa, nasopharyngeal tonsil or soft palate tonsil. Toll-like receptor (TLR)-2 and TLR-9 mRNA expression in the tonsils was significantly increased following B. subtilis administration compared with the control group (P<0.05). The results demonstrate that B. subtilis administration increases the number of immune cells in the nasal mucosa and tonsils of piglets and stimulates nasal mucosal and tonsillar immunity. The present study lays the foundation for further study into the intranasal administration of B. subtilis in humans to enhance the immunity of human nasal mucosa to respiratory diseases.