In 30-50% of coronary artery disease patients receiving saphenous vein grafts or bare-metal stents reoccurrence of symptoms (restenosis) occurs due to intimal thickening involving vascular smooth muscle cell (VSMC) proliferation. By investigating regulators of VSMC proliferation we aim to reduce restenosis. The Proline-Rich Homeodomain protein (PRH/Hhex) is a negative regulator of cellular growth in hematopoietic and several other cell types and modulates Wnt/β-catenin signalling through more than one mechanism. We have shown Wnt/β-catenin signalling regulates VSMC proliferation and intimal thickening. This study aimed to determine whether PRH regulates VSMC proliferation and Wnt/β-catenin signalling. Western blotting revealed that induction of proliferation with foetal calf serum (FCS) significantly attenuated PRH protein levels in human saphenous vein VSMCs (0.39±0.04 vs.0.63±0.09, n=3, p<0.05 paired-Student's t-test). Similar results were observed when proliferation was induced with platelet-derived growth factor and basic fibroblast growth factor in both human saphenous vein and rat aortic VSMCs. MG132 revealed the loss of PRH protein was due to enhanced proteosomal degradation. Using AMAXA nucleofection of specific siRNA, PRH mRNA and protein levels were significantly reduced (20±12% and 60±10% n=3, p<0.05 Student's t-test, respectively), and proliferation was significantly enhanced (21±3 vs. 12±2% proliferation, n=3, p<0.05 paired-Student's t-test) in human VSMCs. We also observed that enhancing PRH protein levels using an adenovirus encoding PRH significantly reduced Wnt/β-catenin signalling to 68±2%, n=3, p<0.05, Student's t-test. In summary, we showed that PRH inhibited VSMC proliferation in part via retarding Wnt/β-catenin signalling. Design of novel treatments to increase PRH levels may be useful for retarding restenosis.