Abstract: 【Objective】 This study aimed to investigate the effects of growth factor receptor binding protein 2，（GRB2） gene expression knockdown on proliferation and apoptosis of porcine mammary epithelial cells， providing references for revealing the biological functions of GRB2 gene in porcine mammary gland development. 【Method】 Four pairs of specific interference targeting sequence （shRNA-GRB2-877， shRNA-GRB2-555， shRNA-GRB2-692， shRNA-GRB2-366） and one pair of control sequence （shRNA-NC） were designed and synthesized based on GRB2 gene reference sequences. The shRNA with the highest inhibition efficiency was screened for TMT-labeled quantitative proteomics analysis. Flow cytome-try was used to detect cell cycle and apoptosis， and the CCK-8 assay was used to measure cell proliferation activity. 【Result】 After GRB2 gene expression knockdown， the relative expression of GRB2 gene in shRNA-GRB2-366， shRNA-GRB2-555， shRNA-GRB2-692， and shRNA-GRB2-877 groups was extremely significantly lower than that in shRNA-GRB2-NC group （P<0.01， the same below）， with shRNA-GRB2-555 showing the highest inhibition efficiency of 62.21%. A total of 250 differentially expressed proteins were identified， in which 53 were up-regulated differentially expressed proteins and 197 were down-regulated differentially expressed proteins. Differentially expressed proteins were predominantly localized to the nucleus and cytoplasm. GO functional annotation analysis showed that the differentially expressed proteins were mainly annotated in functional terms such as immune system process， defense response to virus， antigen processing and presentation， antigen binding， receptor ligand activity， receptor regulator activity， MCH protein complex， and MHC class Ⅰ protein complex. KEGG signaling pathway enrichment analysis revealed that the differentially expressed proteins that significantly enriched in the top 20 signaling pathways were mainly enriched in viral protein interaction with cytokine and cytokine receptor， IL-17 signaling pathway， and NOD-like receptor signaling pathway. shRNA-GRB2-555 interference could induce porcine mammary epithelial cell cycle to arrest at the G1 phase （P<0.05）. Apoptosis rate in shRNA-GRB2-555 group was extremely higher than that in shRNA-NC group， and the proliferation activity at 12， 24， 48， and 72 h after transfection with the interference vector was significantly lower than that of shRNA-NC group. 【Conclusion】 GRB2 gene is the key functional gene in regulating proliferation and apoptosis of porcine mammary epithelial cells. GRB2 gene expression knockdown can extremely significantly increase the proportion of cells at G1 phase， extremely significantly decrease the proportion of cell at S and G2 phases， induce porcine mammary epithelial cell cycle to arrest at G1 phase， and extremely significantly inhibit cell proliferation activity and promote apoptosis.