To increase isobutanol production in Saccharomyces cerevisiae, the valine biosynthetic pathway was activated by overexpression of the relevant enzymes in the mitochondria and the cytosol. Native mitochondrial enzymes were overepxressed in the cytosol by deleting the mitochondrial transit peptides. The metabolically engineered S. cerevisiae possessing the cytosolic pathway showed increased isobutanol production (63 ± 4 mg/L).