Objective: To observe the effects of Exendin-4, a glucagon-like peptide-1(GLP-1) receptor agonist, on proliferation and osteogenic differentiation of Mc3t3-E1(mouse embryonic osteoblast precursor cells) in vitro. Methods: Mc3t3-E1 cells were treated with Exendin-4 at a pre-experimental concentration of 30 nmol/L for 72 h. The cells were collected for sequencing, and the RNA-SEQ data were obtained. The differentially expressed genes greater than 2 times were screened and KEGG PATHWAY enrichment analysis and GO enrichment analysis were performed. Results: Mc3t3-E1 cells were proliferated after Exendin-4 treatment, and a total of 418 differentially expressed genes were screened, including 236 up-regulated genes and 182 down-regulated genes. GO enrichment analysis showed that the up-regulated differentially expressed genes were mainly enriched in the immune system, cellular adhesion and proteolytic pathways. KEGG PATHWAY enrichment analysis showed that The down-regulated differentially expressed genes were mainly enriched in PI3K-Akt signaling pathway, abnormal transcription in cancer, mammalian target of Rapamycin (mTOR) receptor signaling pathway, and hypoxia inducible factor 1 (HYPO xia inducible) Factor-1, HIF-1) signaling pathway. It revealed that up-regulated genes related to osteoporosis included Fosl1, Cxcl13, Clec11a, Phex, Fasl, Camk4, Stab1, Zbp1, Adora2a, Igfbp7, Cxcr2, Hp, MTI; while for Down-regulated genes included Nog, Zeb1, Esr1, Igf2bp2, Plxnb3, Rgs14. Conclusions: Exendin-4 can affect the differentiation of precursor cells into osteoblasts by influencing the expression levels of gene related to cell immunity, cell adhesion and proteolysis related genes, and ultimately affect the formation and development of osteoporosis.