Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD+-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP+ as the cofactor without a significant increase in catalysis using NAD+ as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for kcat,NAD, suggesting that His at residue 362 may be more beneficial than Gln for NAD+ binding. Furthermore, the S346I/K347D/K362H mutant had a very large Km,NADP value compared to other mutants, suggesting that this mutant exclusively utilizes NAD+ as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their Km,NAD values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the Km values for NAD+ and NADP+ of the quadruple mutants were significantly decreased, while either kcat,NAD or kcat,NADP was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD+-utilizing enzymes by a considerable increase in catalysis using NAD+ as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding affinity of NAD+. Here, we demonstrate that a series of E314A-containing c-NADP-ME quadruple mutants have been changed to NAD+-utilizing enzymes by abrogating NADP+ binding and increasing NAD+ binding.