During bone remodeling, there is requirement ofdifferentiation of osteoblastic cells. Previously, we identifiedproteins differentially expressed in soft tissue during bonehealing. Of these proteins, we focused the effect of LTF ondifferentiation of osteoblast. In order to analyze theosteogenic ability of LTF, we treated conditioned mediacollected from human LTF-stably transfected HEK293Tcells into osteoblastic MC3T3-E1. The results showed thatthe activity and expression of alkaline phosphatase wereincreased in MC3T3-E1 cells treated with conditioned mediacontaining LTF in dose- and time-dependent manner. At thesame time, we observed the significant increase of theexpression of osteoblastic genes, such as ALP, BSP,COL1A1, and OCN, and along with matrix mineralizationgenes, such as DMP1 and DMP2, in LTF conditionedmedia-treated groups. Moreover, the result of treatingrecombinant human LTF directly into osteoblasticMC3T3-E1 showed the same pattern of treating conditionedmedia containing LTF. Our study demonstrated that LTFconstitutively enhances osteoblastic differentiation via induction of osteoblastic genes and activation of matrix mineralization in MC3T3-E1 cells.