Clostridium acetobutylicum is able to use a variety of carbohydrates for the production of biotechnological relevant compounds like biobutanol. Here, the utilization of fructose by C. acetobutylicum was investigated. Fructose uptake was found to be mediated by three PTS systems: PTSFru, PTSManI, PTSManII. The three PTS-deficient strains C. acetobutylicum ∆fru, C. acetobutylicum manI::int and C. acetobutylicum ∆manII were constructed to elucidate their function during fructose uptake. Batch fermentations revealed that PTSFru is the primary uptake system for fructose. Its deletion caused significant inhibition of growth, accompanied by a loss of solvent production. Furthermore, the uptake and utilization of fructose by PTSFru is dependent on the regulator FruR. Its deletion caused a decrease in fructose uptake, suggesting this regulator is needed for activation of PTSFru.