Abstract: Hyriopsis cumingii is a unique freshwater pearl mussel in our country. It occupies an important position in the aquaculture industry. In the actual breeding process, male individuals have a more obvious advantage in pearl production than female individuals. Therefore, research on the sex determination of H. cumingii is crucial. Previous studies have found that Sox9 gene plays a role in male sex determination in many species, kinase X gene is an important gene in the synthesis of protein kinase (PKA), and PKA plays a vital role in many physiological and biochemical processes, especially in the process of spermatogenesis. In this process, PKA has a very obvious effect on sperm capacitation. The kinase X gene and Sox9 gene are both located in cAMP-PKA pathway. And the relationship of the kinase X gene and Sox9 gene is upstream and downstream. It is speculated that the Sox9 gene may be regulated by PKA. The results showed that the kinase X gene was 1 652 bp in length, the 3′ UTR was 314 bp, while the length of the 5′ UTR was 45 bp, encoding 430 amino acids, and there was an STKYc structure domain which was located at 381-1179 bp, kinase X gene has a transmembrane domain and the Molecular weight (Mw) of the kinase X protein was 49.00 ku and the theoretical Isoelectric point (pI) was 8.22, among which Leu(L) was the most abundant amino acid, accounting for 9.8%; the homology analysis showed that H. cumingii had a high similarity with other shellfishes such as Mizuhopecten yessoensis, Chlamys farreri. The phylogenetic tree showed that H. cumingii had a close relationship with Crassocras gigas and Crassostrea virginica, but had a long genetic distance with mammals. The fluorescence quantitative PCR results showed that the kinase X gene was expressed in 6 tissues including gonads, gill, adductor muscle, foot, mantle, liver of the 2-year-old H. cumingii , and the kinase X gene was expressed the highest in the male gonads while expressed the lowest in the female liver. And in 1-3 years of H. cumingii, the expression of kinase X gene in male was all higher than that in female, and the difference between male and female was extremely significant. Among them, the expression of kinase X gene in 2-year-old H. cumingii was highest among 1-3 years. Due to the high expression of the kinase X gene of the 2-year-old H. cumingii near the sexual maturity stage, the sperm occurrence rate was high; after 7 days of interference, the expression of kinase X gene decreased. RNA interference results show that the synthesized 3 interference chains had a certain interference efficiency on the kinase X gene, and the interference rate of the interference chain 1 was the highest. The interference rate was 83.1% in females and 81.9% in males, while interference chain 2 had an interference rate of 76.0% in females and 25.7% in males; interference chain 3 had an interference rate of 24.0% in females and 67.7% in males. At the same time, the Sox9 gene expression decreased by 90.3% in females and 56.6% in males after the interference of interfere chain 1. This improved the synthesis of kinase X gene was positively correlated with the synthesis of Sox9 gene. It was speculated that the two may work together to participate in the sex determination process through the cAMP-PKA-mediated signaling pathway. In this experiment, through preliminary functional exploration of the kinase X gene, it was found that there was the upstream and downstream relationship between kinase X gene and Sox9 gene and the two were positively correlated, and it effectively interfered with the kinase X gene, which is important in cAPM-PKA of H. cumingii. This study provides a theoretical basis for the study of sex determination and male parthenogenesis of H. cumingii.