The rhamnose-GlcNAc disaccharide linker is fundamental to the structural integrity of mycobacterial cell wall. The donor dTDP-rhamnose is synthesized by four enzymes (RmlA, B, C, and D) beginning with dTTP and glucose-1-phosphate. We generated M. smegmatis rmlB gene knockout mutant (transcription of downstream rmlC gene was blocked because of a polar effect) by homologous recombination. When the Mycobacterium tuberculosis (Tb) rmlB rescue plasmid carrying a temperature-sensitive replication origin and Tb rmlC bearing plasmid with a normal replication origin were present in the mc(2)155 rmlB knockout mutant, the mutant was unable to grow at non-permissive temperature (42 degrees C) where the Tb rmlB rescue plasmid is lost. While the Tb rmlC rescue plasmid carrying a temperature-sensitive replication origin and Tb rmlB bearing plasmid with a normal replication origin were present in the mc(2)155 rmlB knock out mutant, this mutant was also unable to grow at the non-permissive temperature where the Tb rmlC rescue plasmid is lost. These results demonstrate that rmlB and rmlC genes are essential for mycobacterial growth, therefore, RmlB and RmlC are essential targets to develop new anti-tuberculosis drugs.