Loss of tissue organization is a hallmark of the early stages of cancer, and there is considerable interest in proteins that maintain normal tissue architecture. Prostate epithelial cells cultured in Matrigel form three-dimensional acini that mimic aspects of prostate gland development. The organization of these structures requires the tumor suppressor Dickkopf-3 (Dkk-3), a divergent member of the Dkk family of secreted Wnt signalling antagonists that is frequently downregulated in prostate cancer. To gain further insight into the function of Dkk-3 in the prostate, we compared the prostates of Dkk3-null mice with those of control littermates. We found increased proliferation of prostate epithelial cells in the mutant mice and changes in prostate tissue organization. Consistent with these observations, cell proliferation was elevated in acini formed by human prostate epithelial cells stably silenced for Dkk-3. Silencing of Dkk-3 increased TGF-?/Smad signalling, and inhibitors of TGF-?/Smad signalling rescued the defective acinar phenotype caused by loss of Dkk-3. These findings suggest that Dkk-3 maintains the structural integrity of the prostate gland by limiting TGF-?/Smad signalling.