The production of biofuels from renewable sources by microbial engineering has gained increased attention due to energy and environmental concerns. Butanol is one of the important gasoline-substitute fuels and can be produced by native microorganism Clostridium acetobutylicum. To develop a fundamental tool to understand C. acetobutylicum, a high resolution proteome reference map for this species has been established. To better understand the relationship between butanol tolerance and butanol yield, we performed a comparative proteomic analysis between the wild-type strain DSM 1731 and its mutant Rh8 at acidogenic and solventogenic phases, respectively. The 102 differentially expressed proteins that are mainly involved in protein folding, solvent formation, amino acid metabolism, protein synthesis, nucleotide metabolism, transport, and others were detected. Hierarchical clustering analysis revealed that over 70% of the 102 differentially expressed proteins in mutant Rh8 were either upregulated (e.g., chaperones and solvent formation related) or downregulated (e.g., amino acid metabolism and protein synthesis related) in both acidogenic and solventogenic phase, which, respectively, are only upregulated or downregulated in solventogenic phase in the wild-type strain.