Abstract: The caspase 3（CASP3） is an influential member of the caspase family（CASP）, which is located downstream of orderly cascade of apoptotic reaction and it is convergence point of the transmission of a variety of apoptotic stimuli. Its activation has also become a sign that apoptosis has entered the irreversible phase. However, the regulatory mechanism of CASP3 gene in the occurrence of sheep mastitis is still unclear. Therefore, this experiment takes Hu sheep as the experimental animal, the CASP3 gene CDS sequence obtained by the transcriptome sequencing in the early stage of the research group was used. The bioinformatics method was used to analyze the physical and chemical properties, structure and homology of CASP3 protein, and focused using RT-qPCR, western blot and immunohistochemistry methods to analyze the expression and localization of CASP3 gene in mammary tissues of hu sheep mastitis. Bioinformatics analysis showed that the Hu sheep CASP3 protein was a stable hydrophilic protein, a non-secreted protein, and did not have a transmembrane region, multiple phosphorylation sites. The evolutionary level was highly conservative during evolution. Moreover, the results of RT-qPCR and western blot showed that CASP3 gene expression was present in healthy and clinical mastitis mammary tissues, and the relative expression of CASP3 mRNA and protein in clinical mastitis mammary tissues was extremely significant（P＜0.01） higher than healthy group, immunohistochemistry showed that CASP3 protein mainly exists in stromal cells of healthy mammary tissues, but it was strongly distributed in clinical mastitis mammary tissues stromal cells and mammary epithelial cells. It is suggested that the expression of CASP3 mRNA and protein are up-regulated in clinical mastitis mammary tissues. This provides basic information for further research on the regulatory role of CASP3 gene in the pathogenesis of Hu sheep mastitis.