Abstract: Artemia sinica is a small crustacean inhabiting extreme environments such as salt lakes and salt pans, which adapts to harsh stresses including low temperature and high salinity by producing diapause embryo. To explore the function and regulatory mechanism of Chloride Channel 3 (CLC3) during the diapause process of Artemia, the CLC3 gene with a total length of 2923 bp was identified and cloned in this study by screening the Artemia transcriptome database. It contains an open reading frame (ORF) of 2376 bp and encodes a protein of 791 amino acids. Quantitative Real-time PCR demonstrated that CLC3 mRNA expression was the lowest in diapause embryo produced by oviparous Artemia relative to other developmental stages. Subsequently, dsRNA-mediated RNA interference was employed to knock down CLC3 expression in ovoviviparous Artemia. Individuals in the experimental group produced diapause embryos, whereas those in the control group released free-swimming nauplii. In addition, the dormancy-specific molecular chaperones p26 and Artemin showed marked up-regulation and accumulation in the experimental group. Collectively, our findings indicate that down-regulating CLC3 expression promotes the formation of diapause embryo, offering a novel perspective for understanding the conserved functions of chloride channel proteins in adaptive dormancy across organisms.