An experimental investigation is conducted to evaluate the effects of using blends of n-butanol (normal butanol) with conventional diesel fuel, with 8% and 16% (by vol.) n-butanol, on the performance and exhaust emissions of a fully instrumented, six-cylinder, water-cooled, turbocharged and after-cooled, heavy duty, direct injection (DI), Mercedes-Benz engine, installed at the authors’ laboratory, which is used to power the mini-bus diesel engines of the Athens Urban Transport Organization sub-fleet. The tests are conducted using each of the above fuel blends, with the engine working at two speeds and three loads. Fuel consumption, exhaust smokiness and exhaust regulated gas emissions such as nitrogen oxides, carbon monoxide and total unburned hydrocarbons are measured. The differences in the measured performance and exhaust emissions of the two butanol/diesel fuel blends from the baseline operation of the engine, i.e. when working with neat diesel fuel, are determined and compared. It is revealed that this fuel, which can be produced from biomass (bio-butanol), is a very promising bio-fuel for diesel engines. The differing physical and chemical properties of n-butanol against those for the diesel fuel, aided by sample cylinder pressure and heat release rate diagrams, are used to interpret the observed engine behavior.