This study examines the proteolytic activity of the kefir grains (a combination of bacteria and yeast) on bovine milk proteins. SDS-PAGE analysis reveals substantial digestion of milk proteins by the kefir grains in comparison with control samples. Mass spectrometric analysis reveals that the kefir microorganisms released 609 new peptide fragments and significantly altered the abundance of around 1,500 peptides compared to the controls. These kefir-digested peptides derived from 55 milk proteins. We show that kefir contains 25 previously identified functional peptides with actions including antihypertensive, antimicrobial, opioid and anti-oxidative .