The mouse MC3T3-E1 osteoblasts were divided into control group， 20 nmol·L^－1 RAPA group， 60 nmol·L^－1 RAPA group， 160 nmol·L^－1 RAPA group and 2.0 μmol·L^－1 MHY1485 group. The percentages MC3T3E1 osteoblasts at different cell cycles in various groups were detected by flow cytometry；the expression levels of mTOR and downstream signals—IF4E-binding protein 1 （4E-BP1） and ribosome protein subunit 6 kinase 1 （S6K1），B cell lymphoma-2（Bcl-2），Bcl-2 related X protein（Bax），and Caspase-3，alkaline phosphatase （ALP）， Runt-related transcription factor 2 （Runx2），and osteoblast-related transcription factor Osterix were detected by Western blotting and Real-time fluorescence quantitative PCR（RT-qPCR） methods；the mineralization ability of MC3T3-E1 osteoblasts in various groups was observed by Alizarin red staining.

Compared with control group， the proliferation activities of MC3T3-E1 osteoblasts in 20 and 60 nmol·L^－1RAPA groups were significantly increased（P<0.05 or P<0.01）， the percentages of MC3T3-E1 osteoblasts at G₁ phase were increased（P<0.05 or P<0.01），the percentages of the MCET3-E1 osteoblasts at S phase were increased（P<0.05 or P<0.01），the expression levels of p-mTOR and its downstream pathway of p-4E-BP1 and p-S6K1， as well as p62，Bax，and Cleaved-Caspase-3 were decreased（P<0.05 or P<0.01），and the expression levels of LC3-Ⅱ， Bcl-2， ALP and Runx2 were increased （P<0.05 or P<0.01）.Compared with control group，the proliferation activity of MC3T3-E1 osteoblasts in 160 nmol·L^－1 RAPA group was significantly decreased（P<0.01），and the expression levels of p-mTOR，p-4E-BP1， and p-S6K1 were decreased （P<0.05 or P<0.01）；the proliferation activity of MC3T3-E1 osteoblasts in 2.0 μmol·L^－1 MHY1485 group was decreased（P<0.01），and the expression levels of p-mTOR，p-4E-BP1， and p-S6K1 were decreased（P<0.01）；however，the expression levels of Bax and Cleaved-Caspase-3 in both 160 nmol·L^－1 RAPA and 2.0 μmol·L^－1 MHY1485 groups were increased（P<0.05 or P<0.01），and the expression levels of Bcl-2， ALP， Runx2， and Osterix were decreased（P<0.05 or P<0.01）.Twelve days after treatment， the mineralized nodules were found in the MC3T3-E1 osteoblasts in 20 and 60 nmol·L^－1RAPA groups， and only a very small amount of calcium salt were found in the MC3T3-E1 osteoblasts in 160 nmol·L^－1 RAPA and 2.0 μmol·L^－1 MHY1485 groups.

Mild to moderate inhibition of mTOR phosphorylation can promote the proliferation， autophagy and differentiation of the MC3T3-E1 osteoblasts，while excessive inhibition and enhancement of mTOR phosphorylation have the opposite effect.