Abstract: Objective To establish the Jurkat T cell line which stably overexpresses or down expresses miR-125b-5p. Methods The plasmid was amplified and packaged into 293T cells to produce lentiviral particles. Jurkat T cells were infected with recombinant lentivirus. The expression level of miR-125b-5p in Jurkat T cells was detected by RT-qPCR. Expression level of target gene UVRAG was detected by Western blotting. All data were analyzed by one-way ANOVA with LSD test as post-hoc test. P＜0.05 was regarded as statistically significant. Results The emition of green fluorescence was detected in both Jurkat T cells that transfected with lentivirus overexpress hsa-miR-125b-5p vector or control vector. Besides, the emition of red fluorescence was detected in both Jurkat T cells that transfected with lentivirus hsa-miR-125b-5p interferential vector and control vector. The RT-qPCR results showed that compared with the control vector group, the expression level of miR-125b-5p was significantly increased in Jurkat T cells which were transfected with hsa-miR-125b-5p lentivirus over expression vector (P<0.01). The expression level of miR-125b-5p was significantly decreased in Jurkat T cells which were transfected with hsa-miR-125b-5p lentivirus interferential vector (P<0.01). Western blotting results showed that compared with the control vector group, the protein level of UVRAG was significantly decreased in Jurkat T cells which were transfected with hsa-miR-125b-5p lentivirus over expression vector (P<0.001). The protein level of UVRAG was significantly increased in Jurkat T cells which were transfected with hsa-miR-125b-5p lentivirus interferential vector (P<0.001). Conclusion Jurkat T cell lines with stable overexpression or low expression of hsa-miR-125b-5p were successfully established.