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This study aimed to analyze metabolite abundances and proteome differences between buffalo milk from Binglangjiang (BBM) and Dehong (DBM). Untargeted UPLC-MS/MS, label-free proteomics approach, and bioinformatics analyses including GO, KEGG, and PPI were performed. The results revealed that catechol, L-proline, γ-aminobutyric acid, hippuric acid, creatinine, and betaine were detected in BBM and DBM as the major metabolites. One hundred thirteen proteins, mainly within the molecular weight range of 20–80 kDa, were identified, and the differential protein numbers of BBM were higher than those of DBM. These proteins were primarily involved in cholesterol metabolism, Staphylococcus aureus and Salmonella infections, the peroxisome proliferator-activated receptor signaling pathway, and coagulation cascades. Relative abundances of α-s1-casein, α-s2-casein, and β-casein were plentiful in BBM, while κ-casein, albumin, and α-lactalbumin were in DBM. PPI network analysis indicated that the identified proteins were highly interconnected. These findings promote a better understanding of quality biomarkers in dairy product improvements. [ABSTRACT FROM AUTHOR]