Sp1 regulates the activation of many genes involved in tumor growth, apoptosis, and angiogenesis. We have previously shown the involvement of Sp1 in the up-regulation of urokinase receptor (uPAR) expression, a key molecule in tumor invasion and metastasis. Here, we investigated whether a marked down-regulation of Sp1 activity may inhibit uPAR expression and migration ability of MDA-MB-231 breast cancer cells. To this end, we tested the decoy ability of a novel peptide nucleic acid (PNA)-DNA chimera which carries a central DNA strand, containing Sp1-binding sequence, covalently linked to two PNA fragments at both ends (PNA-DNA-PNA, PDP). The chimera was synthesized, annealed with complementary DNA (PDP-DNA), and then tested for its ability to bind Sp1 both in vitro and in living MDA-MB-231 breast cancer cells in the presence of urokinase (uPA). This PDP-DNA decoy molecule efficiently competes for the binding to endogenous Sp1 in nuclear extracts, and upon transfection with liposomal vectors, causes a marked decrease of available Sp1 in both untreated and uPA-treated MDA-MB-231 cells. Accordingly, both uPA-dependent enhancement of uPAR expression and cell migration were strongly reduced in transfected cells. Interestingly, a detectable inhibitory effect is also observed in breast cancer cells exposed to PDP-DNA in the absence of transfection reagents. Finally, the inhibitory effect of PDP-DNA appeared to be stronger than that observed with oligonucleotides carrying Sp1 consensus sequence. Our findings show that this novel PNA-DNA chimera, containing Sp1 consensus sequence, effectively inhibits Sp1 activity, uPAR expression, and motility of breast cancer cells indicating its potential therapeutic use to prevent tumor dissemination.