Mesenchymal cells in the liver provide the microenvironment for hepatoblasts expansion and differentiation. We have previously demonstrated that myofibroblasts (MFs) promoted hepatoblasts differentiation into cholangiocytes, whereas its role in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes remains elusive. Here, we investigated the role of MFs in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes using an indirect coculture system. When cocultured with hepatoblasts, MFs promoted hepatoblasts differentiation into cholangiocytes and inhibited the proliferation and stemness of hepatoblasts. However, when hepatoblasts already differentiated into cholangiocytes, MFs promoted the differentiated cholangiocytes proliferation. In addition, hepatoblast proliferation genes such as hepatocyte growth factor (HGF), insulin-like growth factor-1 and 2 (IGF-1 and 2), midkine 1 (Mdk1), and pleiotrophin (Ptn) expression in MFs were down-regulated compared with their levels in fibroblasts. Our findings uncover the role of MFs in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes, potentially providing a novel therapeutic strategy for cholangiocyte regeneration. (C) 2019 Published by Elsevier Inc.