Background and aims: Repeated applications of glyphosate in short timeframes can lead to periodic accumulation of soilborne glyphosate residues. This study aimed to determine whether typical glyphosate residues observed in fields affect growth, plant metabolite composition and fungal root endophyte colonisation of wheat (Triticum aestivum). Methods: Glyphosate was applied to a sandy soil at 0, 0.33, 1, 3, 9 and 27 times a recommended label rate (LR) to establish soil glyphosate concentrations of 0, 0.1, 0.4, 1, 4.7 and 14.8 mg kg−1 in topsoil (0–100 mm) after a 28 d plant-free incubation. Wheat plants were then grown to establish thresholds of soilborne glyphosate on seedling emergence, major alterations to plant metabolic responses and fungal root endophyte colonisation. Results: Seedling emergence was unaffected by soil residual glyphosate treatments. Shoot biomass was significantly higher at 1 mg kg−1 compared to the control treatments (P < 0.05) and root biomass followed a similar trend. Leaf metabolite profiles of plants growing in soil containing 4.7–14.8 mg kg−1 glyphosate could be discriminated from the 0–0.4 mg kg−1 treatments due to significant (P < 0.05) effects on the relative concentrations of metabolites. Fungal root endophyte colonisation was significantly reduced to 10% at 14.8 mg kg−1 (P < 0.05) whereas shoot and root biomass remained unaffected. Conclusions: Our study highlights differences in glyphosate sensitivity thresholds between plants and fungal symbionts. Sub-lethal concentrations of residual soilborne glyphosate can alter wheat metabolism and impair fungal root endophyte colonisation. Effect thresholds for glyphosate may be lower for fungal symbionts than for host plants. However, the soil glyphosate residue levels required to reach such thresholds are high and would only be approached in commercial fields with a history of repeated glyphosate application.