Intranasal immunization with whole inactivated virus (WIV) is an important strategy used for influenza prevention and control. However, a powerful mucosal adjuvant is required to improve nasal vaccine efficacy. Riboflavin, as a food additive with the advantages of being safe and low-cost, widely exists in living organisms. In this paper, the mucosal adjuvant function of riboflavin was studied. After intranasal immunization with H1N1 WIV plus riboflavin in mice, we found that the mucosal immunity based on the secretory IgA (sIgA) levels in the nasal cavity, trachea, and lung were strongly enhanced compared with H1N1 WIV alone. Meanwhile, the IgG, IgG1, and IgG2a levels in serum also showed a high upregulation and a decreased ratio of IgG1/IgG2a, which implied a bias in the cellular immune response. Moreover, riboflavin strongly improved the protection level of H1N1 inactivated vaccine from a lethal influenza challenge. Furthermore, riboflavin was found to possess the capacity to induce dendritic cell (DC) phenotypic (MHCII, CD40, CD80, and CD86) and functional maturation, including cytokine secretion (TNF-α, IL-1β, IL-12p70, and IL-10) and the proliferation of allogeneic T cells. Lastly, we found that the DC maturation induced by riboflavin was dependent on the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which plays an important role in immune regulation. Therefore, riboflavin is expected to be developed as an alternative mucosal adjuvant for influenza nasal vaccine application.