We have hypothesized that epithelial growth, branching, and canalization in the rodent ventral prostate (VP) would require matrix remodeling, and hence matrix metalloproteinase (MMP) activity. Therefore, the aim of this study was to evaluate the impact of blocking MMP-2, using whole organ culture. siRNA was employed to inhibit MMP-2 expression, and this was compared to GM6001's (a broad-spectrum MMP inhibitor) inhibition of general MMPs. These blocks impaired VP morphogenesis. MMP-2 silencing reduced organ size, epithelial area, and the number of tips, as well as caused a dilation of the distal parts of the epithelium. Histology, 3-D reconstruction, biochemistry, and second harmonic generation (SHG) revealed that MMP-2 silencing affected VP architecture by interfering in epithelial cell proliferation, lumen formation, and cellular organization of both epithelium and stroma, besides intense accumulation of collagen fibers. These data suggest that MMP-2 plays important roles in prostate growth, being directly involved with epithelial morphogenesis.