Objective To establish and characterize GSK-3β knockout thymic epithelial cells in miceand evaluate their immunological characteristics. Methods The Cre-LoxP system was used for the F0 generation of GSK-3βflox/ flox(GSK-3βf/ f ) mice with targeted gene knockout in embryonic stem cells. PCR was used to analyze GSK-3βf/ f FoxN1-Cre+/ -(GSK-3β-/ - ) mice with specific knockout of the GSK-3β gene in thymus epithelial cells. The general biological characteristics, reproductive ability, and progeny survival rate of gene knockout mice were observed. HE staining, flow cytometry, and immunofluorescence were used to compare the immune organ structure, thymus, spleen, and proportion and proliferation of immune cells in peripheral blood between knockout and wildtype mice. Results Mice with specific knockout of the GSK-3β gene in thymus epithelial cells were established successfully. Compared with wildtype mice, GSK-3β-/ - mice showed no significant difference in general biological characteristics, and the survival rate of offspring was > 90%. In the aging process, the thymus of knockout mice was larger than that of wildtype mice with a higher thymus index and more thymic cells. The numbers of initial T cells and recently migrated cells in the spleen were significantly higher than those in wildtype mice. Conclusions Mice with specific knockout of the GSK-3β gene in thymic epithelial cells was established successfully. These mice provide a good tool to study the function and mechanism of the GSK-3β gene in thymus epithelial cells.