Abstract: 【Objective】The aim of this study was to screen，identify and analyze the functions of cellular proteins interacting with annexin A2（ANXA2）in chicken theca cells，which provided theoretical basis for further studying the mechanism of ANXA2 regulating chicken follicular development.【Method】The chicken theca cells of 30-week-old Guizhou yellow laying hens were prepared and then used to extract the total proteins. The chicken ANXA2 recombinant protein His-ANXA2 and His tag protein were used as bait proteins，respectively，to screen the cellular proteins interacting with His-ANXA2 and His by His Pull-Down combined with mass-spectrometric technique（LC-MS/MS）method. The GO function annotation，KEGG signaling pathway and protein-protein interaction network analysis of the identified proteins was performed using the bioinformatics online software. Protein interaction networks were drawn using STRING Version 11.0.【Result】A total of 41 cellular proteins that interacted with chicken ANXA2 were identified by His Pull-Down combined with LC-MS/MS. GO annotation analysis showed that these interacting cellular proteins played roles in molecular function，biological processes，and cell composition. As for the molecular function analysis，these proteins were involved in protein binding（58.06%），catalytic activity（19.35%），structural consituent of ribosome（16.13%），and structural constitute of cytoskeleton（6.45%）. The results of biological processes analysis revealed that these proteins mainly participated in cytoskeleton organization（19.35%），to stimulus response（19.35%），translation（16.13%），metabolic process （12.90%），cell migration（12.90%），protein folding（9.68%），and protein transport（9.68%）. However，the distribution of cell membrane（32.26%）was the main localization pattern in cell composition analysis. Meanwhile，KEGG signaling pathway analysis showed that these proteins were related to stress response，metabolism，translation，signal transduction， immune system，and protein localization. The protein-protein interaction networks were divided into three ways，including CNN2-FN1-MYH9-MYH10-ACTN1-CSRP1，ANXA1-ANXA2-ENO1-PRDX4-GPI-ATP5B-PRDX3-HSPA8-TUBB2A， CCT7-CCT4 -GNB2L1-ATP5A1-RPS3-RPS3A-RPL23A-RPL22-RPS7，which showed the complex interactions among them. Of which annexin A1（ANXA1）and enolase -1（ENO1）had the most obvious interactions with ANXA2.【Conclusion】The identified cellular proteins interacting with chicken ANXA2 are mainly involved in the biological processes of cytoskeleton formation，response to stimulationand translation，and the signaling pathways of stress response，metabolism，translation，signal transduction，immunity and protein localization. PRDX3，PRDX4，MYH9，and TCSC possibly participate in the occurrence of chicken ovaries related diseases by interacting with ANXA2，while ANXA1 interacting with ANXA2 might regulate the follicular development and ovulation in chickens.