Glyphosate-based herbicides (GBHs) are used worldwide for weed management. However, GBHs pose a threat to soil fungal community, although fungi can degrade and use glyphosate as a nutrient source. How fungi respond to GBHs remains enigmatic. Here, we found that, not as in plants, the commercial GBH Roundup does not target the 5-enolpyruvyl-shikimate-3-phosphate synthase in the soil-derived fungus