Objective To study the effect of muscle segment homeodomain homeobox 2 (MSX2) on the expression of enamel matrix protein and the formation of enamel. Methods Immunohistochemical staining was used to detect the expression of MSX2 in mouse tooth embryos and its localization in ameloblasts. The short hairpin RNA (shRNA) of the MSX2 gene was designed and synthesized, and then the annealed double stranded DNA was constructed into the pGMLV-SC5 RNAi lentivirus vector, and finally it was packaged with lentivirus. The lentivirus was used to infect ameloblasts. Real-time fluorescent quantitative PCR was performed to screen the best interference fragment, and detect the mRNAs of amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam), amelotin (Amtn) and kallikrein 4 (Klk4). The embryos were isolated for 18.5 days and then infected with RNAi recombinant lentivirus targeting MSX2. The tooth germ was implanted under the renal capsule of the mouse. Ten weeks later, the tissue was harvested to separate and observe the tooth form and contour. Results MSX2 was expressed in the secretory phase and maturation phase of mouse ameloblasts, but the expression signal was weaker in the secretory phase and was stronger in the mature stage. The lentivirus of MSX2-shRNA targeting MSX2 gene we constructed inhibited the expression of Amelx and Klk4 mRNAs. The RNAi lentivirus targeting MSX2 gene infected the tooth enamel and led to a decrease in the degree of enamel mineralization. Conclusion The MSX2 gene is expressed in ameloblasts. The knockdown of MSX2 can inhibit the expression of enamel matrix proteins and the enamel mineralization.