Metabolic engineering has been used to improve L-ornithine biosynthesis in Escherichia coli W3110. L-Ornithine production increased from 0.3 to 3.2 mg/g (dry cell weight) when the primary L-ornithine biosynthetic pathway was optimized by disrupting the pathway transcription repressor, thereby increasing the expression of the genes involved in the pathway, and by preventing conversion of L-ornithine into citrulline. When a feedback-resistant N-acetylglutamate synthetase gene (argA214) was placed under the control of the arabinose-inducible promoter, either in the chromosome or on a multicopy plasmid in the cell, the combination of overexpression of argA214 with an argF argI argR triple knockout mutation had an additive effect on L-ornithine production but only when exogenous glutamate was present. When speF (which encodes ornithine decarboxylase) and proB (which encodes gamma-glutamyl kinase) were inactivated to prevent the conversion of L-ornithine to putrescine and to block the biosynthesis of a side branch of L-ornithine, respectively, L-ornithine production was further enhanced approxi. 140% from 5.5 to 13.2 mg/g (dry cell weight).