Poly-gamma-glutamic acid (γ-PGA), an extracellular biopolymer produced by Bacillus sp., is a non-canonical toll-like receptor 4 (TLR4) agonist. Here we show its antiviral efficacy against noroviruses. γ-PGA with a molecular mass of 2,000-kDa limited murine norovirus (MNV) replication in the macrophage cell line RAW264.7 by inducing interferon (IFN)-β and conferred resistance to viral infection-induced cell death. Additionally, γ-PGA interfered with viral entry into cells. The potent antiviral state mounted by γ-PGA was not attributed to the upregulation of TLR4 or TLR3, a sensor known to recognize norovirus RNA. γ-PGA sensing by TLR4 required the two TLR4-associated accessory factors MD2 and CD14. In ex vivo cultures of mouse ileum, γ-PGA selectively increased the expression of IFN-β in villi. In contrast, IFN-β induction was negligible in the ileal Peyer's patches (PPs) where its expression was primarily induced by the replication of MNV. Oral administration of γ-PGA, which increased serum IFN-β levels without inducing proinflammatory cytokines, reduced MNV loads in the ileum with PPs and mesenteric lymph nodes in mice. Our results disclose a γ-PGA-mediated non-conventional TLR4 signaling in the ileum, highlighting the potential use of γ-PGA as a prophylactic antiviral agent against noroviruses.