The present invention provides a method of producing butanol, comprising: causing a microorganism which belongs to the genus Clostridium and is capable of producing butanol to produce butanol in a medium comprising saccharides assimilable by a microorganism which belongs to the genus Clostridium, lactic acid and/or acetic acid as substrates. By the method of the present invention, the concentration of butanol produced in a culture medium (solution) can be increased and, furthermore, the rate of butanol production can also be increased. In the present invention, a mixture of D-lactic acid and L-lactic acid obtained by lactic acid fermentation or a fermentation product of hetero-lactic acid fermentation containing acetic acid as a by-product can be utilized. By combining lactic acid fermentation and the method of the present invention, a practical system of butanol production from inedible biomass which requires neither expensive enzyme nor advanced technology such as genetic manipulation can be constructed.