Lactoferrin (LF) is known to modulate the bone anabolic effect. Previously, we and others reported that the effects of LF on the bone may be conferred by the stimulation of transforming growth factor β (TGF-β) signaling in the preosteoblast. However, the underlying molecular mechanisms of LF-induced osteogenic differentiation of mesenchymal stem cells (MSCs) has not been identified. In this study, we tested the hypothesis that the effects of LF on osteogenesis of MSCs required mediation by TGF-β Receptors and activating TGF-β signaling pathway. Using siRNA silencing technology, the knockdown of TGF-β Receptor II (TβRII) could significantly attenuate LF's effect on the proliferation rate and alkaline phosphatase (ALP) activity of MSCs. It indicated that LF induced osteogenic activity that is dependent on TβRII in C3H10T1/2. Subsequently, it was shown that LF activated Smad2. Downregulating TGF-β Receptor I (TβRI) with SB431542 attenuated the expression of p-Smad2 and p-P38, also the LF-induced the osteogenic activity. Besides, the stimulation by LF on the expression of Osteocalcin (OCN), Osteopontin (OPN), Collagen-2a1 (Col2a1), and Fibroblast Growth Factor 2 (FGF2) were abolished by SB431542. These results confirmed that LF induced osteogenic activity though the TGF-β canonical and noncanonical signaling pathway. This study provided the first evidence of the signaling mechanisms of LF's effect on osteogenesis in MSCs.