Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol producing strain. However, little information is available concerning its butanol production mechanism and the development of more robust strains. In this study, key biosynthetic genes (either endogenous or exogenous) including the sol operon (bld-ctfA-ctfB-adc), adhE1, adhE1(D485G), thl, thlA1(V5A), thlA(V5A) and the expression cassette EC (thl-hbd-crt-bcd) were over-expressed in C. saccharoperbutylacetonicum N1-4 to evaluate their potential in enhancement of butanol production. The overexpression of sol operon increased ethanol production by 400%. The overexpression of adhE1 and adhE(D485 G) resulted in a 5.6- and 4.9-fold higher ethanol production, respectively, producing final acetone-butanol-ethanol (ABE) titers (30.6 and 30.1 g L-1) of among the highest as ever reported for solventogenic clostridia. The most significant increase of butanol production (by 13.7%) and selectivity (73.7%) was achieved by the overexpression of EC. These results provides a solid foundation and essential references for the further development of more robust strains.