Proline (abbreviated as Pro or P) is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary. The more common L form has S stereochemistry.

Proline is biosynthetically derived from the amino acid L-glutamate and its immediate precursor is the imino acid (S)-1-pyrroline-5-carboxylate (P5C). Enzymes involved in a typical biosynthesis include:

The distinctive cyclic structure of proline's side chain locks its φ backbone dihedral angle at approximately −60°, giving proline an exceptional conformational rigidity compared to other amino acids. Hence, proline loses less conformational entropy upon folding, which may account for its higher prevalence in the proteins of thermophilic organisms. Proline acts as a structural disruptor in the middle of regular secondary structure elements such as alpha helices and beta sheets; however, proline is commonly found as the first residue of an alpha helix and also in the edge strands of beta sheets. Proline is also commonly found in turns, which may account for the curious fact that proline is usually solvent-exposed, despite having a completely aliphatic side chain. Because proline lacks a hydrogen on the amide group, it cannot act as a hydrogen bond donor, only as a hydrogen bond acceptor. The distinct side chain/amine group interactions allow proline to aid in the formation of beta turns and also explains why it is not apparent in alpha helices.