Proline, a unique proteogenic secondary amino acid, has its own metabolic system with special features. Recent findings defining the regulation of this system led us to propose that proline is a stress substrate in the microenvironment of inflammation and tumorigenesis. The criteria for proline as a stress substrate are: 1) the enzymes utilizing proline respond to stress signaling; 2) there is a large, mobilizable pool of proline; and 3) the metabolism of proline serves special stress functions. Studies show that the proline-utilizing enzyme, proline oxidase (POX)/proline dehydrogenase (PRODH), responds to genotoxic, inflammatory, and nutrient stress. Proline as substrate is stored as collagen in extracellular matrix, connective tissue, and bone and it is rapidly released from this reservoir by the sequential action of matrix metalloproteinases, peptidases, and prolidase. Special functions include the use of proline by POX/PRODH to generate superoxide radicals that initiate apoptosis by intrinsic and extrinsic pathways. Under conditions of nutrient stress, proline is an energy source. It provides carbons for the tricarboxylic acid cycle and also participates in the proline cycle. The latter, catalyzed by mitochondrial POX and cytosolic pyrroline-5-carboxylate reductase, shuttles reducing potential from the pentose phosphate pathway into mitochondria to generate ATP and oxidizing potential to activate the cytosolic pentose phosphate pathway.