In order to investigate the role of the KLHL10 gene in the sex development of Hyriopsis cumingii, the full length of its cDNA was cloned using RACE, and KLHL10 expression levels in six tissues (gonad, gill, adductor muscle, foot, mantle, and hepatopancreas) were determined. Further, the quantitative real-time PCR was performed to compare expressions levels between 1-8 months of age and 12-, 24-, and 36-month-old H. cumingii, and its function was explored using RNA interference. The results showed that the full length of the KLHL10 gene was 2 361 bp, and the 5' non-coding region was 93 bp, the 3' non-coding region was 447 bp, and the open reading frame was 1 821 bp which encoded 606 amino acids. qRT-PCR results showed that the gene was highly expressed in the testis, and was expressed at the highest level at 6 months of age in early stage. Moreover, in 12-, 24-, and 36-month-old individuals, KLHL10 gene expression levels in the testis were higher than those in the ovaries(P<0.05). At the same time, the double-stranded RNA (dsRNA) interference experiments were also performed using three dsRNA strands. The results showed that the expression of KLHL10 in gonad was effectively reduced. According to the above results, it is speculated that the KLHL10 gene is a male-related gene in H. cumingii, which may participate in sex differentiation and testis development of H. cumingii.