It is well known that bovine milk contains gamma-glutamyltransferase (GGT) activity. To verify the identity of the GGT and further to characterize the generation of gamma-glutamyl peptides, identification of GGT from bovine milk and quantification of kokumi peptides and free amino acids were performed. GGT was purified from skim milk and identified as the bovine protein (G3N2D8), and it reveals that it is composed of two subunits. Sequence alignment with human GGT and molecular mass determination showed that the bovine GGT was glycosylated and contained an N-terminal transmembrane part. Further activity characterization was performed in comparison with GGT from Bacillus amyloliquefaciens in terms of the ability to generate gamma-glutamyl peptides from casein hydrolysates. During the transpeptidation reaction catalyzed by both GGT, gamma-glutamyl peptides significantly (P < 0.05) increased after gamma-glutamylation; addition of glutamine contributed to the generation of gamma-glutamyl peptides, suggesting that glutamine could act as a gamma-glutamyl donor. This study reveals that the GGT of skim milk membranes is a glycosylated membrane protein that can generate gamma-glutamyl peptides.