The myeloid differentiation factor 2 (MD-2)–related lipid-recognition (ML) domain is found in multiple proteins, including MD-2, MD-1, Niemann–Pick disease type C2, and mite major allergen proteins. The significance of ML proteins in antibacterial signal transduction and in lipid metabolism has been well studied. However, their function in host–virus interaction remains poorly understood. In the current study, we found that the ML protein family is involved in resistance against white spot syndrome virus in kuruma shrimp, Marsupenaeus japonicus. One member, which showed a high similarity to mammalian MD-2/MD-1 and was designated as ML1, participated in the antiviral response by recognizing cholesta-3, 5-diene (CD), a lipid component of the white spot syndrome virus envelope. After recognizing CD, ML1 induced the translocation of Rel family NF-κB transcription factor Dorsal into the nucleus, resulting in the expression of Vago, an IFN-like antiviral cytokine in arthropods. Overall, this study revealed the significance of an MD-2 homologue as an immune recognition protein for virus lipids. The identification and characterization of CD–ML1–Dorsal–Vago signaling provided new insights into invertebrate antiviral immunity.