Abstract: 【Objective】The aim of this study was to investigate the inhibitory effects of hesperidin on apoptosis of bovine mammary epithelial cells and its molecular mechanism,so as to provide a theoretical basis for its application in promoting the growth and development of mammary gland and maintaining lactation function in cows.【Method】Apoptosis model of bovine mammary epithelial cells was generated by treating it with 1000μmol/L H₂O₂ for 8 h,and then treated with 120μg/mL hesperidin for 24 h.Cell viability was measured by CCK-8 assay,and apoptosis was detected by confocal microscopy,flow cytometer and DNA gel electrophoresis.The expression levels of Bcl-2,Bax and Caspase3 genes were measured by real-time fluorescence quantitative PCR and the protein expression levels of Bcl-2,Bax and Caspase3 were measured by Western blotting.【Result】Compared with the blank control group(CK),the bovine mammary epithelial cell viability was decreased extremely significantly (P<0.01,the same below) and the apoptosis proportion was extremely significantly increased in H₂O₂ group,with the DNA fragments appearing as a ladder pattern;the bovine mammary epithelial cell viability was improved significantly in hesperidin group (P<0.05,the same below),while apoptosis rate was decreased and the number of apoptotic cells was lower in hesperidin group.Compared with H₂O₂group,the bovine mammary epithelial cell viability was significantly increased and apoptosis rate was extremely significantly decreased,and DNA ladder fragments were decreased in H₂O₂+hesperidin group.In terms of genes expression of Bax,Bcl-2,and Caspase3 and proteins expression,in H₂O₂ group,Bax and Caspase3 genes expression and protein expression levels were significantly up-regulated,while Bcl-2 gene expression and its protein expression levels were down-regulated compared with CK group.In hesperidin group,Bax and Caspase3 gene expression and their protein expression levels were significantly or extremely significantly down-regulated compared with CK,Bcl-2 gene expression and its protein expression levels were up-regulated compared with CK group.Compared with H₂O₂ group,Bax and Caspase3 gene expression and their proteins expression in H₂O₂+hesperidin group were extremely significantly down-regulated,and Bcl-2 gene expression and its protein expression were extremely significantly up-regulated.【Conclusion】Hesperidin inhibits H₂O₂-induced apoptosis of bovine mammary epithelial cells and effectively enhances cell viability through Bcl-2/Bax-Caspase3 pathway,it is speculated that hesperidin has a good positive effect on promoting mammary organ development and maintaining lactation abilityin dairy cow.