Arabinosyltransferases are a family of membrane-bound glycosyltransferases involved in the biosynthesis of the arabinan segment of two key glycoconjugates, arabinogalactan and lipoarabinomannan, in the mycobacterial cell wall. All arabinosyl-transferases identified have been found to be essential for the growth of Mycobcterium tuberculosis and are potential targets for developing new antituberculosis drugs. Technical bottlenecks in designing enzyme assays for screening for inhibitors of these enzymes are (1) the enzymes are membrane proteins and refractory to isolation; and (2) the sole arabinose donor, decaprenylphosphoryl-d-arabinofuranose is sparingly produced and difficult to isolate, and commercial substrates are not available. In this study, we have synthesized several analogues of decaprenylphosphoryl-d-arabinofuranose by varying the chain length and investigated their arabinofuranose (Araf) donating capacity. In parallel, an essential arabinosyltransferase (AftC), an enzyme that introduces α-(1→3) branch points in the internal arabinan domain in both arabinogalactan and lipoarabinomannan synthesis, has been expressed, solubilized, and purified for the first time. More importantly, it has been shown that the AftC is active only when reconstituted in a proteoliposome using mycobacterial phospholipids and has a preference for diacylated phosphatidylinositoldimannoside (Ac2PIM2), a major cell wall associated glycolipid. α-(1→3) branched arabinans were generated when AftC–liposome complex was used in assays with the (Z,Z)-farnesylphosphoryl d-arabinose linear α-d-Araf-(1→5)3–5 oligosaccharide acceptors and not with the acceptor that had a α-(1→3) branch point preintroduced.