Diabetes mellitus is a common endocrinopathy in cats; it is associated with lesions in the pancreatic islets e.g. islet amyloidosis and beta cell loss. Research on isolated islets greatly contributed to the understanding of the pathophysiology of diabetes in humans. Therefore, we aimed at improving the existing methods of isolation in order to increase islet yield, purity, viability and functionality of isolated islets in cats. Nine cats were used as donors and islet isolation was successfully accomplished in eight cases. The isolation method that achieved the highest islet quality was characterized by the perfusion of the pancreas with 80ml of Collagenase type IV (Worthington) through the pancreatic duct at the site of the major papilla. The enzymatic digestion of the intact organ was combined with mechanical disruption and controlled by frequent dithizone staining. Purification was performed by a first filtration step followed by handpicking. Purified islets were then plated on extracellular matrix pre-coated plates (ECM) plates and cultured for 48h before a functionality test was performed. For the first time, it was possible to isolate and culture feline islets with high degree of viability and purity. However, as the islet yield and the percentage of islet free from the acinar tissue relative to the total number of isolated islets was low compared to data on other species, further studies are required to improve the procedure of islet isolation in cats.