The nasal mucosa is composed of multiple layers of barrier structures and is the first line of defense against infection by respiratory pathogenic microorganisms. A large number of commensal microorganisms are present in the nasal mucosa that mediate and regulate nasal mucosal barrier function. The objective of this research was to investigate the effects of commensal microorganisms on the nasal mucosal barrier. The results revealed that the strain of Bacillus velezensis (B. velezensis) NSV2 from the nasal cavity has good probiotic abilities to resist Pasteurella multocida, Staphylococcus aureus, Escherichia coli and Salmonella typhimurium. Lambs were subsequently administered intranasally with B. velezensis NSV2 at 3, 12, 21, and 26 days old, respectively. For the microbial barrier, although B. velezensis NSV2 reduces the diversity of nasal microbiota, it significantly increased the relative abundance of beneficial bacteria in the nasal cavity, and reduced the abundance of potential pathogenic bacteria. For the mucus barrier, the number of goblet cells in the nasal mucosa significantly increased after B. velezensis NSV2 treatment. For the immune barrier, B. velezensis NSV2 also significantly increased the number of IgA B cells, CD3 T cells and dendritic cells in the nasal mucosa, as well as the mRNA expression of interleukin (IL) 6, IL11, CCL2, and CCL20 (P < 0.05). The protein level of CCL20 also significantly raised in nasal washings (P < 0.05). Moreover, the heat-inactivated and culture products of B. velezensis NSV2 also drastically induced the expression of CCL20 in nasal mucosa explants (P < 0.05), but lower than that of the live bacteria. This study demonstrated that a symbiotic strain of B. velezensis NSV2 could improve the nasal mucosal barrier, and emphasized the important role of nasal symbiotic microbiota.