The retinoblastoma (Rb)-E2F transcriptional regulatory pathway plays a major role in cell-cycle regulation, but its role in invasion and metastasis is less well understood. We find that many genes involved in the invasion of cancer cells, such as matrix metalloproteinases (MMP), have potential E2F-binding sites in their promoters. E2F-binding sites were predicted on all 23 human MMP gene promoters, many of which harbored multiple E2F-binding sites. Studies presented here show that MMP genes such as MMP9, MMP14, and MMP15 which are overexpressed in non-small cell lung cancer, have multiple E2F-binding sites and are regulated by the Rb-E2F pathway. Chromatin immunoprecipitation assays showed the association of E2F1 with the MMP9, MMP14, and MMP15 promoters, and transient transfection experiments showed that these promoters are E2F responsive. Correspondingly, depletion of E2F family members by RNA interference techniques reduced the expression of these genes with a corresponding reduction in collagen degradation activity. Furthermore, activating Rb by inhibiting the interaction of Raf-1 with Rb by using the Rb-Raf-1 disruptor RRD-251 was sufficient to inhibit MMP transcription. This led to reduced invasion and migration of cancer cells in vitro and metastatic foci development in a tail vein lung metastasis model in mice. These results suggest that E2F transcription factors may play a role in promoting metastasis through regulation of MMP genes and that targeting the Rb-Raf-1 interaction is a promising approach for the treatment of metastatic disease.