DEAD (Asp-Glu-Ala-Asp)-box polypeptide 41 (DDX41), a receptor belonging to the DExD family, has recently been identified as an intracellular DNA sensor in vertebrates. Here, we report on the identification and functional characterization of PmDDX41, the first cytosolic DNA sensor in shrimp. By searching a Penaeus monodon expressed sequence tag (EST) database (http://pmonodon.biotec.or.th), three cDNA fragments exhibiting similarity to DDX41 in various species were identified and assembled, resulting in a complete open reading frame of PmDDX41 that contains 1863-bp and encodes a putative protein of 620 amino acids. PmDDX41 shares 83% and 79% similarity to DDX41 homolog from the bee Apis florea and fruit fly Drosophila melanogaster, respectively and contains three conserved domains in the protein: DEADc domain, HELICc domain, and zinc finger domain. The transcript of PmDDX41 was detected in all tested tissues and was up-regulated upon infection with a DNA virus, white spot syndrome virus (WSSV). However, PmDDX41 mRNA expression was not significantly changed and down-regulated in response to a bacterium, Vibrio harveyi, or an RNA virus, yellow head virus (YHV), respectively, compared with the control phosphate-buffered saline-injected shrimp. Furthermore, the suppression of PmDDX41 by dsRNA-mediated gene silencing resulted in more rapid death of WSSV-infected shrimp and a significant decrease in the mRNA expression levels of several immune-related genes (PmIKKβ, PmIKKε, PmRelish, PmCactus, PmDorsal, PmPEN3, PmPEN5, and ALFPm6). These results suggest that PmDDX41 is involved in the antiviral response, probably via a DNA-sensing pathway that is triggered through the IκB kinase complex and leads to the activation of several immune-related genes.