The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the L-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved L-ornithine production by Corynebacterium glutamicum. Production of L-ornithine requires 2 moles of NADPH per mole of L-ornithine. Thus, the effect of NADPH availability on L-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of L-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum ΔAPRE::rocG, produced 14.84 g l⁻¹ of L-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.