The antiproliferative activity of transforming growth factor-β (TGF-β) is essential for maintaining normal tissue homeostasis and is lost in many types of tumors. Gene responses that are central to the TGF-β cytostatic program include activation of the cyclin-dependent kinase inhibitors, p15(Ink4B) and p21(WAF1/Cip1), and repression of c-myc. These gene responses are tightly regulated by a repertoire of transcription factors that include Smad proteins and Sp1. The DLX4 homeobox patterning gene encodes a transcription factor that is absent from most normal adult tissues, but is expressed in a wide variety of malignancies, including lung, breast, prostate and ovarian cancers. In this study, we demonstrate that DLX4 blocks the antiproliferative effect of TGF-β. DLX4 inhibited TGF-β-mediated induction of p15(Ink4B) and p21(WAF1/Cip1) expression. DLX4 bound and prevented Smad4 from forming complexes with Smad2 and Smad3, but not with Sp1. However, DLX4 also bound and inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc independently of TGF-β/Smad signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-β cytostatic program could explain, in part, the resistance of tumors to the antiproliferative effect of TGF-β.