Diabetes Mellitus is by far one of the most propagated chronic diseases, affecting 150 million people worldwide. This affliction is caused by a malfunction of pancreatic endocrine cells, which provokes a failure in the insulin release and glucose homeostasis. Plasma membrane KATP channels have a key role in the stimulus-secretion coupling of pancreatic β-cells. Consequently, many investigations have developed efficient drugs for the treatment of diabetes, such as sulphonylureas, which specifically close KATP channels leading to an enhanced insulin secretion. Recent studies show that, in addition to its well-known plasma membrane location, sulphonylurea receptors and sulphonylurea-sensitive KATP channels are also present in various intracellular sites including secretory granules, mitochondria, endoplasmic reticulum and more recently, the nucleus. What roles do they play in these organelles? Intracellular KATP channels and sulphonylurea receptors, which operate in conjunction with classical pathways, can provide specific signaling circuits to establish direct links between extracellular signals and different cell functions, such as secretion or gene expression. The study of these intracellular channels provides novel perspectives in the signal transduction of the pancreatic β-cell, and may offer clues for the development of new strategies in diabetes therapy. In this review we will address this topic with special emphasis on the biophysical basis and functional implications in the pancreatic β-cell.