Fungus garden enzyme activity is crucial for sustaining societies of attine ants. The evolutionary diversification of this clade has likely been influenced by enzymatic specialization in connection to changes in foraging niche, particularly when the ancestral leaf-cutting ants shifted from a diet of mostly fresh but shed plant material to actively cutting leaves. However, the way in which these ants managed to overcome the chemical defences of leaves has remained poorly understood. Here we document that fungal laccases may have played an important role in allowing the leaf-cutting ants to become generalist functional herbivores. Laccases are polyphenol oxidase enzymes (PPOs) that are best known for their ability to degrade lignin in saprophytic and wood-pathogenic fungi. We found that laccase activity was primarily expressed in newly constructed garden sections where secondary leaf compounds are most likely to hinder decomposition. A combination of genomic and transcriptional analyses showed that there are at least eight copies of putative laccase coding genes in a draft genome of the fungal symbiont Leucocoprinus gongylophorus, but only a single copy of these multiple laccase genes is differentially expressed in the modified hyphal tips (gongylidia) that the fungal symbiont produces. We can also show that this laccase enzyme passes through the ant gut to be expressed in the fecal droplets that the ants mix with their chewed-up fresh leaf forage, providing strong indications for an adaptive function of this enzyme in the ant-fungus symbiosis. We hypothesize that fungus garden laccases originally had a lignin-degrading function in the fungi that the attine ants domesticated, consistent with the gardens of lower attine ants processing only dry plant substrate. Our results would then suggest that a single of these laccases genes was co-opted for the derived function of neutralizing secondary phenolic defences of leaves. Selection for increased gene expression of fungal laccase genes in the ancestral leaf-cutting ant symbiont could thus have gradually increased the ant’s capacity to process a higher proportion of fresh leaves in their forage, facilitating the evolutionary transition to a leaf-cutting life style.