The arabinogalactan (AG) of Corynebacterianeae is a critical macromolecule that tethers mycolic acids to peptidoglycan, thus forming a highly impermeable cell wall matrix termed the mycolyl-arabinogalactan peptidoglycan complex (mAGP). The front line anti-tuberculosis drug, ethambutol (Emb), targets the Mycobacterium tuberculosis and Corynebacterium glutamicum arabinofuranosyltransferase Mt-EmbA, Mt-EmbB and Cg-Emb enzymes, respectively, which are responsible for the biosynthesis of the arabinan domain of AG. The substrate utilized by these important glycosyltransferases, decaprenylmonophosphoryl-D-arabinose (DPA), is synthesized via a decaprenylphosphoryl-5-phosphoribose (DPPR) synthase (UbiA), which catalyzes the transfer of 5-phospho-ribofuranose-pyrophosphate (pRpp) to decaprenol phosphate to form DPPR. Glycosyl compositional analysis of cell walls extracted from a C. glutamicum::ubiA mutant revealed a galactan core consisting of alternating β(1→5)-Galf and β(1→6)-Galf residues, completely devoid of arabinan and a concomitant loss of cell-wall-bound mycolic acids. In addition, in vitro assays demonstrated a complete loss of arabinofuranosyltransferase activity and DPA biosynthesis in the C. glutamicum::ubiA mutant when supplemented with p[14C]Rpp, the precursor of DPA. Interestingly, in vitro arabinofuranosyltransferase activity was restored in the C. glutamicum::ubiA mutant when supplemented with exogenous DP[14C]A substrate, and C. glutamicum strains deficient in ubiA, emb, and aftA all exhibited different levels of DPA biosynthesis.