The epidemic expansion of diabetes is a major concern of public health. A promising treatment is the transplantation of islets of Langerhans isolated from the whole pancreas but the yields of islets isolation and the rates of successful engraftments still have to be improved to make this therapy effective. The extracellular matrix (ECM) of the pancreatic tissue is partially lost during the isolation process and a comprehensive knowledge of the pancreatic ECM composition and organization could identify targets to improve islets isolation and transplantation or highlight new therapeutics for pancreatic diseases. The organization, composition and three-dimensional architecture of the pancreatic ECM were analysed in mouse and pig by three different techniques. Laminin α-4 and β-2 chains are localized by immunohistochemistry in the exocrine tissue and inside islets of mouse pancreas but not around islets that are surrounded by an ECM made of collagen type IV and type V. Collagen type I, III, and VI were identified by proteomics as specific constituents of the pig pancreatic ECM along with the low-abundance isoforms α3(IV) α4(IV) α5(IV) and α1(V) α2(V) α3(V) of collagen type IV and type V respectively. The three-dimensional ECM architecture is analysed on decellularized mouse pancreas by scanning electron microscopy and is organized in honeycomb structures made of thin ECM fibers assembled in thicker bundles. The combination of immunohistochemistry, proteomics and scanning electron microscopy gives complementary perspective on the pancreatic ECM composition and organization. It represents a valuable toolbox for deeper investigations of ECMs and proposes clues in tissue engineering of the pancreas.