The connecting peptide, or C-peptide, is a short 31-amino-acid polypeptide that connects insulin’s A-chain to its B-chain in the proinsulin molecule. In the insulin synthesis pathway, first preproinsulin is translocated into the endoplasmic reticulum of beta cells of the pancreas with an A-chain, a C-peptide, a B-chain, and a signal sequence. The signal sequence is cleaved from the N-terminus of the peptide by a signal peptidase, leaving proinsulin. After proinsulin is packaged into vesicles in the Golgi apparatus (beta-granules), the C-peptide is removed, leaving the A-chain and B-chain, bound together by disulfide bonds, that constitute the insulin molecule.

Patients with diabetes may have their C-peptide levels measured as a means of distinguishing type 1 diabetes from type 2 diabetes or Maturity onset diabetes of the young (MODY).[11] Measuring C-peptide can help to determine how much of their own natural insulin a person is producing as C-peptide is secreted in equimolar amounts to insulin. C-peptide may be used for determining the possibility of gastrinomas associated with Multiple Endocrine Neoplasm syndromes (MEN 1). Since a significant number of gastrinomas are associated with MEN involving other hormone producing organs (pancreas, parathyroids, and pituitary), higher levels of C-peptide together with the presence of a gastrinoma suggest that organs besides the stomach may harbor neoplasms. C-peptide levels may be checked in women with Polycystic Ovarian Syndrome (PCOS) to help determine degree of insulin resistance.