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Background: Xyloglucan is an important component in plant cell walls that herbivores cannot digest without microbial symbionts. Leaf-cutting ants are major insect herbivores in the Neo-Tropics that rely on fungus-garden enzymes for degrading plant cell walls. However, many of these ants discard much of their harvested plant material after partial degradation, which has led to the hypothesis that the fungal symbionts are primarily producing cell wall degrading enzymes to gain access to intracellular nutrients rather than for obtaining sugars from recalcitrant cell wall polymers, such as (hemi-)cellulose. Results: The fungal symbiont provides a single xyloglucanase (Xeg1) to its ant farmers by upregulating the expression of this protein in the inflated hyphal tips (gongylidia) that the ants ingest. Similar to other enzymes ingested this way, also Xeg1 is not digested but vectored to the fresh leaf-fragment pulp at the top of fungus gardens via ant fecal fluid. Xeg1 is 4-5 times more active in fecal fluid when ants ingest their normal fungal food, compared to a sucrose control diet, as expected when they cannot produce Xeg1 themselves. We confirm substrate specificity of fungal Xeg1 towards xyloglucan by heterologous expression in yeast and show that xyloglucanase activity is higher in the oldest, bottom layers of fungus gardens and in discarded debris material than in the upper and middle layers of fungus gardens. Conclusion: Our results are consistent with Xeg1 playing a role in the initial breakdown of plant cell wall hemicellulose to provide sugars for aggressive hyphal growth before intracellular proteins become available. Xeg1 does not play a major decomposition role in the middle layer of fungus gardens where it is produced by the gongylidia. Overall high xyloglucanase activity in old mycelium that is (about to be) discarded is striking and quite possibly serves defensive purposes by precluding that competing microorganisms can grow. Our results support the hypothesis that the ant-fungus symbiosis prioritizes access to the protein-rich contents of live plant cells and that carbohydrates are not a limiting resource. [ABSTRACT FROM AUTHOR]