The whey acidic protein (WAP) domain is characterized by a 'four-disulfide-core' (4-DSC) motif comprising of approximately 50 amino acids with eight highly conserved cysteine residues. Previous research indicated that WAP domain-containing proteins played an important role in the innate immunity of crustaceans. In the present study, a novel double WAP domain (DWD)-containing protein gene was identified from Chinese mitten crab Eriocheir sinensis (designated EsDWD) by expressed sequence tag (EST) analysis and PCR techniques. The full-length cDNA of EsDWD was of 593 bp, consisting of a 5'-terminal untranslated region (UTR) of 71 bp, a 3' UTR of 120 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 402 bp. The ORF encoded a polypeptide of 133 amino acids with the predicted molecular weight of 14.4 kDa and the theoretical isoelectric point of 8.14, including a signal peptide of 22 amino acids and two WAP domains. The EsDWD mRNA transcripts were ubiquitously expressed in all the tested tissues, and its expression level in gill was significantly higher than that in other tissues. The mRNA expression of EsDWD in haemocytes was up-regulated after challenge of Vibrio anguillarum and Pichia pastoris GS115, as well as injury treatment. The cDNA encoding the mature EsDWD protein was cloned and expressed in Escherichia coli BL21 (DE3) pLysS, and the purified recombinant EsDWD (rEsDWD) protein exhibited antimicrobial activities against Gram-negative bacteria V. anguillarum, yeast P. pastoris GS115 and Candida parapsilosis. The results collectively suggested that EsDWD was a novel member of double WAP domain (DWD)-containing proteins, and involved in the immune defense against microorganism and wound healing in E. sinensis.