Abstract: By this invention, for the first time, a method for high-efficiency genetic transformation of the anaerobic bacterium Clostridium pasteurianum is provided. Clostridium pasteurianum is a bacterium of substantial industrial importance, due to its selectivity and high productivity of the biofuel and biochemical n-butanol, and its ability to grow on a wide variety of inexpensive substrates. Notable among the substrates that it can utilize as a sole source of carbon and energy is glycerine, which is produced in increasing quantities globally as a by-product of biodiesel processing. The industrial exploitation of Clostridium pasteurianum has previously been impeded by the lack of genetic engineering tools for this bacterium. This invention provides such tools for the first time. Included in the invention is a means for protecting newly introduced DNA from degradation by a restriction enzyme within C. pasteurianum. Then, a detailed protocol is given, which enables high-efficiency transformation of C.