Objective：This study aims to explore the function of CASK gene on the regulation of diabetes mellitus in successfully constructed and identified mice model by conditionally CASK knockout in islet β-cells. Methods：CASKloxp/- female mice and CASKloxp/Y male mice were crossed to obtain CASKloxp/loxp female mice and CASKloxp/Y male mice，and then the male mice with conditioned islet beta cells expressing Cre recombinase and CASKloxp/loxp female mice were crossed to obtain CASKloxP/YMIP-Cre male mice and CASKloxP/-MIP-Cre female mice. Mice with CASKloxP/YMIP-Cre genotype are the model mice needed for this experiment. Mice were tailed 1-2 weeks after birth，and their genotypes were identified by PCR. The expression of Cre recombinase was induced by intraperitoneal injection of tamoxifen at 4-5 weeks. Real-time fluorescence quantitative PCR and Western blot were used to verify the knockout effect of CASK gene. Results：Twenty-eight male mice with CASKloxP/YMIP-Cre genotype were obtained after 10-month cross- breeding. Genotype validated by PCR analysis were CASKloxP/YMIP-Cre. The expression of CASK in islets decreased significantly in CASKloxP/YMIP-Cre mice detected by real-time fluorescence quantitative PCR and Western blot. Conclusion：By using Cre-loxp recombination system，the mice model with conditionally CASK deleted in islets was successfully constructed，which provided a research platform for studying the role of CASK gene in the pathogenesis of diabetes mellitus.