Proline racemase (PrdF), which is important for energy metabolism via the Stickland pathway and is unique to certain clostridia, was investigated as a potential anti-Clostridium difficile target by examining its effects on the growth and virulence of C. difficile. Inactivation of PrdF by insertional mutagenesis did not affect early logarithmic growth but only attenuated growth in the mid- and late logarithmic phases. There was no effect on virulence in vivo, suggesting that PrdF is also not required for C. difficile infection. These findings indicate that PrdF as well as other enzymes encoded by the proline reductase operon are all nonessential and are unsuitable targets for anti-C. difficile drug discovery.