Many bacteria require L-rhamnose as a key cell wall component. This sugar is transferred to the cell wall using an activated donor dTDP-L-rhamnose, which is produced by the dTDP-L-rhamnose biosynthetic pathway. We determined the crystal structure of the second enzyme of this pathway dTDP-alpha-D-glucose 4,6-dehydratase (RfbB) from Bacillus anthracis. Interestingly, RfbB only crystallized in the presence of the third enzyme of the pathway RfbC; however, RfbC was not present in the crystal. Our work represents the first complete structural characterization of the four proteins of this pathway in a single Gram-positive bacterium.