Activation of hepatic progenitor cells (HPCs) is commonly observed in chronic liver disease and Wnt/β-catenin signaling plays a crucial role in the expansion of HPCs. However, the molecular mechanisms that regulate the activation of Wnt/β-catenin signaling in the liver, especially in HPCs, remain largely elusive. Here, we reported that ectopic expression of Smad6 suppressed the proliferation and self-renewal of WB-F344 cells, a HPC cell line. Mechanistically, we found that Smad6 inhibited Wnt/β-catenin signaling through promoting the interaction of C-terminal binding protein (CtBP) with β-catenin/T-cell factor (TCF) complex to inhibit β-catenin mediated transcriptional activation in WB-F344 cells. We used siRNA targeting β-catenin to demonstrate that Wnt/β-catenin signaling was required for the proliferation and self-renewal of HPCs. Taken together, these results suggest that Smad6 is a regulatory molecule which regulates the proliferation, self-renewal and Wnt/β-catenin signaling in HPCs. J. Cell. Physiol. 229: 651–660, 2014. © 2013 Wiley Periodicals, Inc.