In this study we introduce a computationally-driven enzyme redesign workflow for altering cofactor specificity from NADPH to NADH. By compiling and comparing data from previous studies involving cofactor switching mutations, we show that their effect cannot be explained as straightforward changes in volume, hydrophobicity, charge, or BLOSUM62 scores of the residues populating the cofactor binding site. Instead, we find that the use of a detailed cofactor binding energy approximation is needed to adequately capture the relative affinity towards different cofactors. The implicit solvation models Generalized Born with molecular volume integration and Generalized Born with simple switching were integrated in the iterative protein redesign and optimization (IPRO) framework to drive the redesign of Candida boidinii xylose reductase (CbXR) to function using the non-native cofactor NADH. We identified 10 variants, out of the 8,000 possible combinations of mutations, that improve the computationally assessed binding affinity for NADH by introducing mutations in the CbXR binding pocket. Experimental testing revealed that seven out of ten possessed significant xylose reductase activity utilizing NADH, with the best experimental design (CbXR-GGD) being 27-fold more active on NADH. The NADPH-dependent activity for eight out of ten predicted designs was either completely abolished or significantly diminished by at least 90%, yielding a greater than 10(4)-fold change in specificity to NADH (CbXR-REG). The remaining two variants (CbXR-RTT and CBXR-EQR) had dual cofactor specificity for both nicotinamide cofactors.