In order to further understand the role of WNT4 in the sex determination process of Hyriopsis cumingii, the full-length cDNA of WNT4 gene was cloned by RACE method(Rapid-amplification of cDNA ends), which was 1 560 bp, including 5'-UTR 396 bp and 3'-UTR 24 bp. The open reading frame (ORF) was 1 140 bp, encoded a putative protein of 379 amino acids that contained a WNT family specific domain. Homology analysis of the amino acid sequence of WNT4 showed that the evolutionary relationship and structural function of the gene were conserved among species. Real-time quantitative PCR (qRT-PCR) analysis of WNT4 gene in the male and female tissues (the mantle, the adductor muscle, the gill, the gonad, the foot and the liver), indicated that it participated in diverse biological processes. It was highly expressed in gill, foot, and liver. There was significant difference between male and female in gonads, adductor muscle, and foot, and the expression of female gonads and adductor muscle was significantly higher than that of males. In situ hybridization results showed that there was obvious hybridization signal in the female oocyte cell. It was speculated that WNT4 gene was related to sex determination.