Scope Kefir is an acidic and alcoholic fermented milk product with multiple health-promoting benefits. Our previous study demonstrated that kefir enhanced calcium absorption in intestinal Caco-2 cells. In this study, kefir-fermented peptide-1 (KFP-1) was isolated from the kefir peptide fraction, and its function as a calcium-binding peptide was characterized. Methods and results KFP-1 was identified as a 17-residue peptide with a sequence identical to that of κ-casein (residues 138-154) in milk protein. KFP-1 was demonstrated to promote calcium influx in Caco-2 and IEC-6 small intestinal cells in a concentration-dependent manner. TRPV6, but not L-type voltage-gated calcium channels, was associated with the calcium influx induced by KFP-1. An in vitro calcium binding assay indicated that the full-length KFP-1 peptide has a higher calcium-binding capacity than the two truncated KFP-1 peptides, KFP-1∆C5 and KFP-1C5. Alexa Fluor 594 labeling showed that KFP-1 is taken up by Caco-2 cells and interacts with calcium ions and TRPV6 protein. Moreover, KFP-1 was found moderately resistant to pepsin and pancreatin digestions and enhanced calcium uptake by intestinal enterocytes in vivo. Conclusion These data suggest that KFP-1, a novel calcium-binding peptide, binds extracellular calcium ions and enters Caco-2 and IEC-6 cells, and promotes calcium uptake through TRPV6 calcium channels. The present study is of great importance for developing kefir-derived metal ion-binding peptides as functional nutraceutical additives. This article is protected by copyright. All rights reserved.