Wnt signaling plays important roles in a variety of developmental and pathological processes. Here we show that Wntless, the main regulator for Wnt secretion, is involved in the innate immune response of the giant freshwater prawn, Macrobrachium rosenbergii. The full-length cDNA of the prawn Wntless (named MrWntless) is 2173 bp in length and contains a 1602-bp open reading frame (ORF), which is conceptually translated into a 533-amino acids sequence. MrWntless protein contains a highly conserved Wnt-binding domain which is required for secretion of Wnt ligands, and exhibits 57–67% identity with known Wntless proteins of other animals. MrWntless was found to be expressed in a variety of prawn tissues including heart, gill, muscle, gut, hepatopancreas and ovary. Moreover, MrWntless expression was significantly increased in the hepatopancreas and gill of the prawns challenged by the bacterial pathogen Aeromonas hydrophila and Vibrio parahaemolyticus. Knockdown of MrWntless by RNA interference in prawns led to dramatically decreased MrWntless expression of approximately 70%. Furthermore, the cumulative mortality rate of the prawn injected with MrWntless dsRNA was greatly increased in response to A. hydrophila challenge compared with the control prawns. Taken together, we provide evidence that prawn Wntless is important for their innate immune response against bacterial pathogens.