The rhamnose-GlcNAc disaccharide is a critical linker which connects arabinogalactan to peptidoglycan via a phosphodiester linkage. The biosynthesis of dTDP-rhamnose is catalysed by four enzymes, and the first reaction is catalysed by an rmlA gene encoding d-glucose-1-phosphate thymidylyltransferase (RmlA). We generated a Mycobacterium smegmatis mc(2)155 mutant lacking the rmlA gene via a homologous recombination method. We tested the requirement for the rmlA gene and the effect of a lack of RmlA on bacterial cell morphology. The results demonstrate that the rmlA gene is essential for mycobacterial growth and that lack of RmlA activity has profound negative effects on bacterial cell morphology. RmlA is thus a potential target for the development of new antituberculosis drugs.