Summary C 4 plants typically operate a CO 2 concentration mechanism from mesophyll (M) cells into bundle sheath (BS) cells. NADH dehydrogenase-like (NDH) complex is enriched in the BS cells of many NADP-ME type C 4 plants, and is more abundant in C 4 than in C 3 plants, but to what extent it is involved in the CO 2 concentration mechanism remains to be experimentally investigated. We created maize and rice mutants deficient in NDH function, and used a combination of transcriptomic, proteomic, and metabolomic approaches for comparative analysis. Considerable decrease in growth, photosynthetic activities, and levels of key photosynthetic proteins were observed in maize but not rice mutants. However, gene expression for many cyclic electron transport and Calvin-Benson cycle components plus BS specific C 4 enzymes, was up-regulated in maize mutants. Metabolite analysis of the maize ndh mutants revealed increased NADPH/NADP ratio, as well as malate, RuBP, FBP, and photorespiration components. We suggest that by optimizing NADPH and malate levels, adjusting NADP-ME activity, NDH functions to balance metabolic and redox states in the BS cells of maize, coordinating photosynthetic gene expression and protein content, thus directly regulating the carbon flow in the two-celled C 4 system of maize.