Xylanases capable of degrading the crystalline microfibrils of 1,3-xylan that reinforce the cell walls of some red and siphonous green algae have not been well studied, yet they could prove to be of great utility in algaculture for the production of food and renewable chemical feedstocks. To gain a better mechanistic understanding of these enzymes, a suite of reagents was synthesized and evaluated as substrates and inhibitors of an endo-1,3-xylanase. With these reagents, a retaining mechanism was confirmed for the xylanase, its catalytic nucleophile identified, and the existence of -3 to +2 substratebinding subsites demonstrated. Protein crystal X-ray diffraction methods provided a high resolution structure of a trapped covalent glycosyl enzyme intermediate, indicating that the 1,3-xylanases likely utilize the S-1(3)-> H-4(3)-> C-4(1) conformational itinerary to effect catalysis.