<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>Isobutanol is an important target for biorefinery research as a next-generation biofuel and a building block for commodity chemical production. Metabolically engineered microbial strains to produce isobutanol have been successfully developed by introducing the Ehrlich pathway into bacterial hosts. Isobutanol-producing baker’s yeast (<jats:italic>Saccharomyces cerevisiae</jats:italic>) strains have been developed following the strategy with respect to its advantageous characteristics for cost-effective isobutanol production. However, the isobutanol yields and titers attained by the developed strains need to be further improved through engineering of <jats:italic>S. cerevisiae</jats:italic> metabolism. </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>Two strategies including eliminating competing pathways and resolving the cofactor imbalance were applied to improve isobutanol production in <jats:italic>S. cerevisiae</jats:italic>. Isobutanol production levels were increased in strains lacking genes encoding members of the pyruvate dehydrogenase complex such as <jats:italic>LPD1</jats:italic>, indicating that the pyruvate supply for isobutanol biosynthesis is competing with acetyl-CoA biosynthesis in mitochondria. Isobutanol production was increased by overexpression of enzymes responsible for transhydrogenase-like shunts such as pyruvate carboxylase, malate dehydrogenase, and malic enzyme. The integration of a single gene deletion <jats:italic>lpd1</jats:italic> Δ and the activation of the transhydrogenase-like shunt further increased isobutanol levels. In a batch fermentation test at the 50-mL scale from 100 g/L glucose using the two integrated strains, the isobutanol titer reached 1.62 ± 0.11 g/L and 1.61 ± 0.03 g/L at 24 h after the start of fermentation, which corresponds to the yield at 0.016 ± 0.001 g/g glucose consumed and 0.016 ± 0.0003 g/g glucose consumed, respectively. </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>These results demonstrate that downregulation of competing pathways and metabolic functions for resolving the cofactor imbalance are promising strategies to construct <jats:italic>S. cerevisiae</jats:italic> strains that effectively produce isobutanol. </jats:sec>