In this paper, we describe a microfabricated cell culture system (MCCS) for the microscopic live observation of cellular behavior in a reconstituted tissue-like microenvironment. The MCCS was used as a novel tool for tracking the proliferation of undifferentiated HT-29 cells. A wall structure of gelled extracellular matrix (ECM) protein was constructed inside the MCCS to establish a tissue-like microenvironment of the intestinal tissue. The ECM wall consisting of type I collagen retained the structural integrity during the gelation process and provided the adhesion surface for the HT-29 cells. We carried out the microscopic live observation of the proliferation of HT-29 cells seeded with various concentrations. The multilayered growth of HT-29 cells in the undifferentiated state was monitored without cell fixation, which was a necessary process in a conventional in vitro cell culture. The live tracking of cell culture also recorded the contraction of cell culture morphology during the initial period of cultivation. This approach is helpful for the establishment of an in vitro model for the live observation study of intestinal epithelial differentiation in an in vivo-like microenvironment.