Mycobacterial cell wall galactan, composed of alternating β-(1→5) and β-(1→6) galactofuranosyl residues, is assembled by the action of two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2, which use UDP-galactofuranose (UDP-Galf) as the donor substrate. Kinetic analysis of synthetic UDP-Galf analogs identified critical interactions involved in donor substrate recognition by GlfT2, a processive polymerizing glycosyltransferase. Testing of methylated UDP-Galf analogs showed the donor substrate-binding pocket is sterically crowded. Evaluation of deoxy UDP-Galf analogs revealed that the C-6 hydroxyl group is not essential for substrate activity, and that interactions with the UDP-Galf C-3 hydroxyl group orient the substrate for turnover but appears to play no role in substrate recognition, making the 3-deoxy-analog a moderate competitive inhibitor of the enzyme. Moreover, the addition of a Galf residue deoxygenated at C-5 or C-6, or an l-arabinofuranose residue, to the growing galactan chain resulted in "dead end" reaction products, which no longer act as an acceptor for the enzyme. This finding shows dual recognition of both the terminal C-5 and C-6 hydroxyl groups of the acceptor substrate are required for GlfT2 activity, which is consistent with a recent model developed based upon a crystal structure of the enzyme. These observations provide insight into specific protein-carbohydrate interactions in the GlfT2 active site and may facilitate the design of future inhibitors.