This study aimed to investigate the role of Pim-1 kinase in the regulation of naïve CD4+T cell differentiation. Naive CD4+T cells were isolated from the spleen of the mice and treated with different cytokines and Pim-1 kinase inhibitor. The expression of T-bet, GATA3, FOXP3 and RORγt proteins in naïve CD4+T cells was detected by Western blot analysis. The expression of cytokines IL-4, TGF-β, IFN- γ and IL-17 in supernatant was detected by ELISA. We found that the inhibition of Pim-1 kinase significantly reduced the expression of T-bet and ROR-γt during naïve CD4+T cells differentiation and inhibited the secretion of IFNγ and IL17 (P<0.05). In conclusion, our study suggests that Pim-1 kinase promotes the differentiation of naive CD4+T cells into Th1 and Th17 cells.