Osteoporosis is a common disease that frequently occurs in the older population, particularly in postmenopausal women. It severely compromises the health of the older population, and the drugs commonly used to treat osteoporosis have a variety of adverse effects. Lactoferrin (LF) is a protein present in milk that has recently been found to exhibit osteogenic activity. Lactoferrin is nontoxic and harmless, suggesting that it may have excellent biocompatibility and tolerability after human consumption. Oral consumption of LF in an ovariectomized rat model has been found to ameliorate osteoporosis. However, the mechanism underlying this effect remains to be clarified. In this study, bovine LF (bLF) was first hydrolyzed by pepsin for 1 h, and the hydrolyzed mixture was freeze-dried and collected. The hydrolyzed mixture was then separated into 5 components (E1-E5), of which E3 had the greatest effect in promoting proliferation of osteoblasts (MC3T3-E1). Component E3 was further isolated into 21 components with preparative reversed phase HPLC, and the E3-15 component had maximal bioactivity. With HPLC-mass spectrometry and peptide sequencing, E3-15 was identified to contain amino acids 97 to 208 from the bLF N terminus. Then, E3-15 was divided into 6 different peptide segments (P1-P6), and the corresponding segments were generated by solid-phase synthesis. Only the P1 peptide (amino acids 97-122 from the N terminus of bLF) significantly promoted osteoblast proliferation. The bioactivity of P1 toward osteoblast cells and alkaline phosphatase activity were tested as a function of P1 concentration, and a nonlinear effect was observed. Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.