Ovotransferrin, the major protein in egg white, is a member of transferrin family. The objective of this study was to study the effects of ovotransferrin on cell proliferation, differentiation, mineralization and osteoclastogenesis of bone osteoblast cells. Effect of ovotransferrin (concentrations ranging from 1 to 1000 μg/mL) on the proliferation, differentiation, and mineralization of mouse osteoblast cells MC3T3-E1 was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay, Western blot, immunofluorescence, and Alizarin-S red staining, respectively. Our results showed that ovotransferrin stimulated cell proliferation (enhanced BrdU incorporation), differentiation (enhanced expression of alkaline phosphatase and type-I collagen), and mineralization (increased calcium deposits) in a dose-dependent manner. Furthermore, ovotransferrin could increase the expression of osteoprotegerin (OPG) while decreasing the expression of receptor activator of nuclear factor kappa-B ligand (RANKL), suggesting its role in inhibition of bone resorption. This study demonstrated for the first time that ovotransferrin might promote bone formation while preventing bone resorption, which might open up a new application of egg white protein ovotransferrin as a functional ingredient in bone health management.