beta-lactoglobulin (beta-lg), a major whey protein was purified and characterised from buffalo colostrum. The in silico analysis of the tryptic peptides based on LC-CID-MS/MS facilitated the identification of protein as beta-lg. The sequences IIVTQ f[1-5] and LSFNPTQLEEQCHV f(149-162) of m/z 933(+) and 851(2+) were found to match N- and C-extreme of beta-lg while IDALNENK f(84-91) and TPEVDDEALEKFDK f(125-138) sequences deduced for m/z 916(+) and 818(2+) were in compliance to buffalo milk beta-lg. Considering the sequence similarity of beta-lg to glycodelin, a proven angiogenic protein, similar role for beta-lg from buffalo colostrum (BLG-col) was examined. Interestingly, BLG-col exhibited anti-angiogenic activity by potently inhibiting cell proliferation, micro-vessel sprouting, cell migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner but having varied effect on Ehrlich ascites tumor cells, MCF-7, MDA-MB 435 and MDA-MB 231 cell lines. The anti-angiogenic potential of BLG-col was found to be vascular endothelial growth factor mediated. The immunolocalisation of BLG-col on the cell surface of HUVECs evidenced using FITC-labelled beta-lg antibody indicated its extra-cellular binding. Furthermore, BLG-col interacting HUVEC membrane protein (64 kDa) was detected by immunoblot and its identity was established by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry analysis, which showed peptide sequence homology to G protein-coupled receptor kinase 4.