Ornithine is a non-proteinogenic α-amino acid that plays a role in the urea cycle. It is not incorporated into proteins during translation. Ornithine is abnormally accumulated in the body in ornithine transcarbamylase deficiency, a disorder of the urea cycle. The moiety derived from ornithine is called ornithyl.

L-Ornithine is one of the products of the action of the enzyme arginase on L-arginine, creating urea. Therefore, ornithine is a central component of the urea cycle, which enables the disposal of excess nitrogen. Ornithine itself is recycled and, in a sense, acts as a catalyst.

First, ammonia is converted into carbamoyl phosphate (H
₂NC(O)OPO²⁻
₃) by carbamoyl phosphate synthetase. Ornithine transcarbamylase then catalyzes the reaction between carbamoyl phosphate and ornithine to form citrulline and phosphate (P_i). Another amino group is contributed by aspartate, leading to the formation of arginine and the byproduct fumarate. The resulting arginine, a guanidinium compound, is subsequently hydrolyzed by arginase to regenerate ornithine and release urea.

The two nitrogen atoms in urea are derived from ammonia and aspartate, while the nitrogen atoms in ornithine remain unchanged.

Ornithine is not an amino acid directly coded for by DNA; that is, it is not a proteinogenic amino acid. However, in mammalian non-hepatic tissues, the primary role of the urea cycle is often the biosynthesis of arginine. As an intermediate in metabolic pathways, ornithine is thus quite important.

Ornithine, through the action of ornithine decarboxylase (EC 4.1.1.17), serves as the starting point for the synthesis of polyamines such as putrescine.

In bacteria such as E. coli, ornithine can be synthesized from L-glutamate.

L-Ornithine supplementation has been shown to attenuate fatigue in subjects in placebo-controlled studies using a cycle ergometer. The results suggest that L-ornithine may exert an antifatigue effect by increasing the efficiency of energy consumption and promoting the excretion of ammonia.