β-Casein phosphopeptides (β-CPP), a class of bioactive peptides derived from β-casein, possess excellent calciumbinding capacity and have potential applications in promoting bone health. In this study, β-CPP was prepared through enzymatic hydrolysis to investigate its effects on the proliferation, differentiation, and mineralization of osteoblasts (MC3T3-E1), with its mechanism of action being analyzed through transcriptomics. MC3T3-E1 cell proliferation rate significantly increased to 128.43% after treating with 50 μg•mL−1 β-CPP for 72 h (P<0.05). The alkaline phosphatase activity and mineralization rate of the β-CPPtreated cells were 2.17 and 2.15 times higher than that of the control cells after 7 and 21 days, respectively, demonstrating that β-CPP significantly enhanced alkaline phosphatase activity and extracellular matrix mineralization in MC3T3-E1 cells (P<0.05). RNA-seq results revealed 476 differentially expressed genes in β-CPP-treated MC3T3-E1 cells, including 258 upregulated and 218 downregulated genes. Gene Ontology analysis indicated that the differentially expressed genes were primarily involved in the regulation of biological processes. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that signaling pathways such as TGF-β and PI3K-Akt were involved in MC3T3-E1 cell differentiation. This study revealed that β-CPP promotes MC3T3-E1 cell proliferation, differentiation, and mineralization, providing a theoretical basis for β-CPP application in bone health and functional foods.