Abstract: 【Objective】 To comprehensively analyze the proteomics and immunohistochemistry-based phosphoproteomics of buffalo testicular spermatogenic cells, and explore the dynamic expression changes of proteins and their regulatory mechanisms during buffalo spermatogenesis, which could provide theoretical basis for analyzing the molecular mechanism of buffalo spermatogenesis. 【Method】 Spermatogenic cells were isolated from testicular tissue of sexually mature buffaloes, and the proteome and phosphorylation proteome expression profiles of buffalo spermatogenic cells were constructed by liquid chromatography-mass spectrometry（LC-MS/MS）. The data were jointly analyzed by bioinformatics softwares to screen out key proteins and their phosphorylation modification sites. Protein interaction networks（PPI） and kinase-substrate interaction networks were constructed. Western blotting and immunohistochemical analysis were used to verify the screened key proteins.【Result】A total of 1785 and 129 proteins were identified in the proteome and phosphoproteome respectively, and 78 shared proteins were identified. High-abundance proteins in the proteome included heat shock proteins and tubulin. GO functional annotation and KEGG signaling pathway enrichment analysis results showed that RNA splicing was significantly enriched in both the proteome and the phosphoproteome（P＜0.05）, and the spliceosome was the signaling pathway with the most phosphorylated proteins enriched. The results of proteomic domain enrichment analysis showed that the RNA recognition motif domain was the most enriched. Cell cycle-dependent protein kinases played a key role in both proteome PPI and kinase-substrate interaction network in phosphoproteome. Among the 78shared proteins identified in the proteome and phosphoproteome, 17 proteins were closely related to spermatogenesis.Western blotting and immunohistochemical analysis showed that both LMNA protein and the phosphorylated form LMNA（pS392） could be successfully identified. LMNA was mainly distributed near the basement membrane of the seminiferous tubules, while LMNA（pS392） was mainly distributed near the lumen of the seminiferous tubules.【Conclusion】 The high expression of heat shock proteins in buffalo testicular spermatogenic cells may be related to the adaptation of buffalo to hot and humid climate, and the high expression of tubulin may be closely related to the migration of spermatogenic cells in seminiferous tubules. RNA splicing and phosphorylation modifications may regulate spermatogenesis by affecting protein function and distribution.