There are increasing demands for the transplantation of tissue-engineered organs. An organ is composed of interconnected networks of ductular structures which transport vital fluids. It is therefore necessary to clarify the controlling factors of three dimensional ductular formation in vitro. Extracellular matrix (ECM) has been reported to be important for ductular formation. Recent studies showed that not only biological signals but also mechanical signals from ECM affected functions and morphology of cells. We hypothesized that the mechanical property of ECM was a controlling factor of three dimensional ductular formation as well. We previously reported that rat biliary epithelial cells (BECs), which constitute bile ducts in liver, reconstructed bile ducts in collagen gel sandwich culture with dimethylsulfoxide (DMSO) stimulation. Here, we investigated the effect of ECM stiffness on ductular formation of BECs. We found that ductular formation of BECs depended on collagen gel stiffness. As collagen gel became stiffer, BECs formed larger bile ducts. Real-time qPCR analysis also revealed that Ki-67 expression was independent of collagen gel stiffness and that Secretin reccptor (SR) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expressions of large bile ducts reconstructed in the stiff collagen gel were higher than those of small bile ducts in the soft collagen gel. These data suggest that enlargement of the bile ducts by the stiff collagen gel is caused by migration of BECs rather than proliferation and that BEC functions depend on the size of reconstructed bile ducts as seen in vivo. In conclusion, we demonstrated that collagen gel stiffness affected the size of the reconstructed bile ducts, which is also leading to the different functions of the bile ducts depending on the size.