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Lymphocyte antigen 6 family member K (LY6K) is upregulated in a number of types of cancer and promotes tumor cell proliferation and metastasis. In addition, LY6K is involved in tamoxifen resistance in breast cancer. However, the in vivo molecular mechanism of LY6K has not yet been investigated. In the present study, transgenic mice overexpressing human LY6K (hLY6K) were generated using the pMAMneo vector, and the effect of LY6K upregulation in vivo was investigated. A total of 4 transgenic mice were generated, and the gene copy number was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RT-qPCR demonstrated that mRNA of hLY6K was overexpressed in the thymus and spleen of the transgenic mice compared with wild-type mice. Flow cytometric analysis demonstrated that the proportions of B and T cells in the spleen were similar in wild-type and transgenic mice; however, the proportion of thymic mature T cells decreased in the transgenic mice, while there was an increase in the proportion of naïve T cells. These findings suggest that the overexpression of LY6K suppresses T cell development, and that LY6K is a potential therapeutic target for cancer.