Urokinase-type plasminogen activator (uPA), expressed in Sertoli cells in the testis, is closely related with tight junctions of blood-testis barrier (BTB), and it has been considered as a potential contraceptive target. In the present study, the antigene effects of triplex-forming oligodeoxynucleotides (TFO) targeting uPA in rat Sertoli cells were investigated in vitro. The stable triplexes, formed by uPA specific TFOs under physiological conditions, were tested by means of electrophoretic mobility shift assays (EMSA). Although tPA, another form of plasminogen activators (PAs), partially compensated the lose of PAs activities, uPA mRNA and protein were significantly reduced as demonstrated by real-time reverse transcription PCR and a chromogenic assay, after the treatment of Sertoli cells with uPA specific TFOs at a concentration of 330 nM. The capacity of TFOs resistance to nuclease degradation was enhanced by the phosphorothioated on the backbone of the oligonucleotides. Our results indicated that the TFOs can downregulate uPA expression and uPA might be an alternative contraceptive target.