<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>L-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering <jats:italic>Corynebacterium glutamicum</jats:italic> to enhance L-ornithine production. </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>A <jats:italic>C. glutamicum</jats:italic> strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved <jats:italic>C. glutamicum</jats:italic> strain, named ΔAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by <jats:italic>C. glutamicum</jats:italic> ΔAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the <jats:italic>ppnK</jats:italic> gene promoted L-ornithine production in <jats:italic>C. glutamicum</jats:italic> ΔAPE6937R42. </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>The availability of NADPH plays an important role in L-ornithine production in <jats:italic>C. glutamicum.</jats:italic> Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds. </jats:sec>