Isobutanol production in Saccharomyces cerevisiae is limited by subcellular compartmentalization of the pathway enzymes. In this study, we improved isobutanol production in S. cerevisiae by constructing an artificial cytosolic isobutanol biosynthetic pathway consisting of AlsS, α-acetolactate synthase from Bacillus subtilis, and two endogenous mitochondrial enzymes, ketol-acid reductoisomerase (Ilv5) and dihydroxy-acid dehydratase (Ilv3), targeted to the cytosol. B. subtilis AlsS was more active than Ilv2ΔN54, an endogenous α-acetolactate synthase targeted to the cytosol. However, overexpression of alsS led to a growth inhibition, which was alleviated by overexpressing ILV5ΔN48 and ILV3ΔN19, encoding the downstream enzymes targeted to the cytosol. Therefore, accumulation of the intermediate α-acetolactate might be toxic to the cells. Based on these findings, we improved isobutanol production by expressing alsS under the control of a copper-inducible CUP1 promoter, and by increasing translational efficiency of the ILV5ΔN48 and ILV3ΔN19 genes by adding Kozak sequence. Furthermore, strains with multi-copy integration of alsS into the delta-sequences were screened based on growth inhibition upon copper-dependent induction of alsS. Next, the ILV5ΔN48 and ILV3ΔN19 genes were integrated into the rDNA sites of the alsS-integrated strain, and the strains with multi-copy integration were screened based on the growth recovery. After optimizing the induction conditions of alsS, the final engineered strain JHY43D24 produced 263.2 mg/L isobutanol, exhibiting about 3.3-fold increase in production compared to a control strain constitutively expressing ILV2ΔN54, ILV5ΔN48, and ILV3ΔN19 on plasmids.