The mechano growth factor (MGF) is originated from alternative splicing of Igf-1 gene, which is mainly expressed in stretched osteoblasts. The main purpose of this study is to examine the effects of MGF and E peptide (the C-terminal 24 amino acids peptide in the E domain of MGF, MGF-Ct24E) on differentiation of osteoblast MC3T3-E1 cells. The results indicate that the MGF and MGF-Ct24E activated the extracellular signal-regulated kinase 1/2(Erk1/2) and affected the expression of osteoblast-associated genes, including the reduced expression of alkaline phosphatase (ALP) and type 1 collagen (Col1), the increased product of osteopontin (OPN) and the decreased nuclear levels of activated Cbfa-1. These effects could be abolished by the blockade of Erk1/2 activation by inhibitor PD98059. In addition, the MGF could preferably activate the PI3K-Akt pathway, which is essential for the differentiation of osteoblasts, so the osteoblast has higher expression of ALP, type 1 Col1, and the formation of bone mineralization nodule under the treatment of MGF than that under the treatment of MGF-Ct24E. These advantages could be inhibited after blockading of Akt by inhibitor LY294002. It was concluded that the MGF-Ct24E could inhibit osteoblasts differentiation through the activated Erk1/2 cascade, while the MGF can induce differentiation through the activated PI3K-Akt. The MGF consists of MGF-Ct24E and IGF-1, which could activate Erk1/2 and PI3K-Akt respectively. Therefore, the MGF possess double effects on differentiation of osteoblasts, which presented as that inhibitory effect at the early differentiation, and promoting effect at the later differentiation.