Norovirus (NoV) infection is a leading cause of gastroenteritis and poses global health threats, with increasing incidence reported in immunocompromised individuals, which is further exacerbated by the globalization of the food industry. Eumelanin has demonstrated its potential in antiviral treatments, but its role in preventing viral infections remains underexplored. Therefore, in this study, we investigated the antiviral properties and potential mechanisms of self-assembled eumelanin nanoparticles (EmNPs) against Tulane virus (TuV), a surrogate with a similar infection mechanism to NoVs. EmNPs exhibited low cytotoxicity and strong antiviral activity in pre-incubated cells. Additionally, EmNPs stimulated the growth and endocytosis of cilia on the cell surface, exposing internal long-nanoparticle chains to interact with the cell membrane while promoting cilia growth and enhancing intercellular connections in cells. EmNPs were then transported to lysosomes via vesicles, leading to a perinuclear lysosome clustering. EmNPs activated several key intracellular signaling pathways, including Toll-like receptor (TLR) and C-type lectin receptor (CLR) pathways, along with activating NF-κB, Rap1, TNF, and Hippo pathways. This regulatory action initiated innate cellular immunity, significantly enhancing the production of type I interferons (IFN-α/β) and promoting the localization of lysosomes to the perinuclear region. Therefore, this study illustrated that EmNPs effectively stimulated immune responses, improved intercellular communication, and facilitated transport mechanisms, thereby bolstering resistance to subsequent viral infections. These findings position EmNPs as promising candidates for the prevention of norovirus infections.