Islet isolation involves enzymatic digestion of the interstitial matrix and mechanical disruption of the tissue. It is possible that a fundamental change of islet biology resulting from the loss of critical factors required for islet function or survival will occur. Extracellular matrix (ECM) is one of the most important components of the islet microenvironment. Reconstruction of the cell-matrix relationship seems to be effective for improving the loss of differentiated islet structure and function. The purpose of this study was to characterize and compare the effects of collagen gel mixture or Matrigel on beta-cell function and islet cell survival. After isolation by the collagenase digestion technique, rat islets were divided and cultured with various types of collagen gel mixture. They were assessed for their glucose-stimulated insulin secretion and cell viability. Glucose-induced insulin secretion of islets cultured with collagen type I gel or a mixture of collagen type I and IV was improved after 11 days in culture. In conclusion, a type of gel composed of collagen type I and/or type IV as an islet microenvironment is sufficient to maintain glucose responsiveness and may be useful for islet transplantation.