The mitogen-activated protein kinase (MAPK) pathway is a conserved signaling system that responds to extracellular signals and translates them into appropriate cellular responses. While multiple MAPK kinase kinases (MAP3Ks) play a crucial role in the step wise transmission of MAPK signals in response to the pathogen infection, little is known about the function of MAP3K15 (also known as apoptosis signal-regulated kinase 3, ASK3) in viral infection. Here, we provide evidence that shrimp MAP3K15 undergoes phosphorylation and activation during a DNA virus, white spot syndrome virus (WSSV) infection, the activated MAP3K15 interacted with the NF-кB homolog, Dorsal, to promote its nuclear translocation for expression of the coiled-coil-containing C-type lectin (CC-CL) and the viral immediate early (ie) genes. CC-CL then activates the JAK/STAT pathway as the ligand to its membrane receptor Domeless, driving the expression of more ie genes. In addition, the JNK/P38 signaling pathway is also activated to promote viral ie genes expression. Importantly, the viral amplification in a wide range of crustaceans were inhibited and the survival rates of host were improved effectively by suppressing MAP3K15 expression or utilizing SDK1, an inhibitor targeting the active form of MAP3K15, suggesting that the MAP3K15 has a critical and conserved function in viral infection. Taken together, this study elucidates a pivotal role and mechanism of MAP3K15 in DNA virus infection, providing novel insights and potential strategies for the control of WSSV infection in crustaceans aquaculture practice.