In mammals and birds, sex is mainly determined by master sex-determination genes on sex chromosomes, which are essential genetic switches controlling sex determination and gonadal differentiation. The sex-determination genes, such as Sry (Sex-determining region Y), Dmrt1 (Doublesex and Mab-3 related transcription factor 1) and DM-W (W-linked DM-domain) genes, are highly conserved in higher vertebrates. In lower vertebrates such as teleost fishes, sex determination can be divided into three categories: genetic sex determination, environmental sex determination and combination of the both. Until now, Dmy/Dmrt1bY (the DM-domain gene on the Y chromosome), Sox3^Y (Sry-related high-mobility group box transcription factor 3 on Y chromosome) and Gsdf^Y (gonadal soma derived growth factor on the Y chromosome) have been identified as Sex-determination genes in fishes. In invertebrates, DM domain-containing genes, such as the Dsx, (doublesex) gene first isolated in Drosophila melanogaster, which was differentially spliced between males and females, are found to be involved in regulating the differentiation of sex-specific cell types. Dmrt1 and other DM-domain containing genes seem to be the major sex-determination genes across metazoan animals. Molluscan reproduction has been an interesting issue in biological research owing to the diverse reproduction strategies evolved in this phylum. In bivalve molluscs, most are dioecism, and some are lifelong hermaphroditic (e.g., bay scallop), and yet some are capable of sex reversal (e.g., Pacific oyster). In addition, the sex of many dioecious bivalves, such as Pinctada fucata and Chlamys nobilis, is not stable and often shows hermaphroditism and sexual reversal. As the importance of sex control in commercial bivalve breeding is concerned, further studies on molecular mechanisms of sex determination and gonad differentiation in molluscs are definitely warranted. To date, there is no clear evidence for the existence of sex chromosomes in bivalves, and whether their sex is controlled by a major gene or multiple genes is still inconclusive. Although some research has been conducted in different bivalves, most have focused on genes homologous to key sex-related genes in model species. For instance, some male-determining genes, including Dmrt, SoxE (Sry-box E), SoxH and Dsx, as well as female-determining genes, including β-catenin, Wnt4 (Wingless-type MMTV integration site family), FoxL2 (forkhead box transcription factor) and its natural antisense transcript FoxL2os, have been reported in some bivalves. Furthermore, genome-wide studies on sex-determining pathways in molluscs are rare due to lack of reference genomes. Dmrt1 is an evolutionarily conserved gene playing vital roles in sex determination from lower vertebrates to mammals. The cDNA sequences of Dmrt1 were obtained from Argopecten irradians irradians by PCR and bioinformatics methods. The expression patterns of AiDmrt1 in different tissues and at different gonadal, embryonic and larval developmental stages were determined by quantitative real-time PCR. Furthermore, the expression characteristics of gonad development-related genes were detected in the AiDmrt1-knockdown testis combined with RNA interference (RNAi) technique. Results showed that the open reading frame of AiDmrt1 was 918 bp encoding 305 amino acids, and the deduced amino acid sequence contained a conserved DM domain. The mRNAs of AiDmrt1 were specifically expressed in testis, reaching a peak value in the testis at growing stage. Compared with fertilized eggs stage, no significant difference in expression levels of AiDmrt1 was observed before the blastula stage, while the expression levels increased significantly at the gastrula stage. When AiDmrt1 was knocked down, testis development-related genes (Sox7 and Sox11) were significantly up-regulated while the expression of Dmrt4 was found to be down-regulated. However, the expression of ovary development-related genes (FoxL2, Wnt4, β-catenin, GATA-1 and GATA-3) was up-regulated significantly. These findings suggested that AiDmrt1 was a testis-specific gene in A. irradians irradians, which was involved in regulation of gonadal development and differentiation in the bay scallop.