Objective: To extract shrimp peptides from the by-products of red shrimp， and study its activity in promoting the proliferation， differentiation and mineralization of MC3T3-E1 cells. Methods: Firstly， the MTT method was used to detect the effect of different concentrations of shrimp peptides on the survival rate of MC3T3-E1 cells. After the concentration was selected， the osteoblasts were induced to differentiate and culture， and the alkaline phosphatase (ALP) activity was measured at 3 and 7 days of culture. Measure the content of osteocalcin (OCN) and type I collagen (COL-I) at 7 d and 14 d. Alizarin red staining is used to detect the degree of mineralization of cells induced and cultured at 21 d. QPCR and Western blot are used to study the expression of key genes and the proteins OPG， RANKL and RUNX2 in bone formation in the OPG/RANKL/RANK signaling pathway. Results: Shrimp peptide mass concentrations of 0.02， 0.05 mg/mL， and 0.1 mg/mL significantly promoted cell proliferation， ALP activity， OCN and COL-I content increased compared to the control group， and the area of mineralized nodules increased significantly (P < 0.05). Shrimp peptide up-regulates the expression of ALP， OCN， and COL-I genes， promotes the expression of OPG and RUNX2 genes and protein levels， and inhibits the expression of RANKL genes and protein levels. Conclusion: Shrimp peptide can promote the proliferation， differentiation and mineralization of MC3T3-E1 osteoblasts， and activate the OPG/RANKL/RANK signaling pathway to promote osteoblast differentiation and bone formation.