The objective of this study is to enhance bio-butanol production using lignocellulosic feedstock via supplements of metabolism perturbation. Metabolic perturbations are non-substrate-based chemical additives that can reinforce metabolic flux towards butanol formation, or increase tolerance to microbial inhibitors in the feedstock. Typical metabolic perturbations include CaCO3, ZnSO4, methyl red, and furan derivatives such as furfural and hydroxymethylfurfural (HMF). In this study, we stepwise tailored metabolic perturbations to maximize butanol production from pure sugar and lignocellulosic feedstock. Under optimized conditions of 4 g/L CaCO3, 2 mg/L ZnSO4, butanol production exceeded 10g/L in wheat straw hydrolysate, which was significantly higher than that obtained in the absent of ZnSO4 and CaCO3. As compared to traditional lignocellulosic feedstock post-treatment method, metabolic perturbations method shows advantages in terms of productivity and economics. Improved bio-butanol production is related to the overexpression of NAD(P)H dependent genes.