Extensive studies have been performed on function of PD-1 in immunological regulation, while as so far, studies on the exact regulation mechanism of PD-1 expression have not been reported. PD-1 expression in EL4 and Sp2/0-Ag cell lines was detected with PE-Anti Mouse-PD-1 antibody through FACS. Genomic DNA from C57BL/6J mouse was used to produce different lengths of PD-1 promoter fragments. The PCR products were cloned into the luciferase reporter vector pGL3-Basic and co-transfected with pRL-SV40 into EL4 and Sp2/0-Ag cell lines to investigate the PD-1 promoter activity. FACS results showed that EL4 cells express PD-1 protein constituently, while Sp2/0-Ag cells express high level of PD-1 after stimulation with phorbol 12-myristate 13-acetate (PMA) plus ionomycin (IO). Luciferase assays revealed that the activity of different lengths of PD-1 promoter region was very alterable. This staggering negative-positive-negative arrangement indicates the complex regulation of PD-1 expression and provides important clues for elucidating the mechanisms of the PD-1 gene transcriptional regulation.