The active herbicide ingredient glyphosate [N-(phosphonomethyl)glycine] is frequently detected as a contaminant in groundwater and surface waters. This study investigated effects of UV-A (365 nm), UV-B (302 nm) and UV-C (254 nm) irradiation of glyphosate in water on photolysis and toxicity to aquatic organisms from different trophic levels. A test battery with bacteria (Bacillus subtilis, Aliivibrio fischeri), a green microalga (Raphidocelis subcapitata), and a crustacean (Daphnia magna) was used to assess biological effect of glyphosate and bioactive transformation products before and after UV irradiation (4.7-70 J/cm2). UV-C irradiation at 20 J/cm2 resulted in a 2-23-fold decrease in toxicity of glyphosate to aquatic test organisms. UV-B irradiation at 70 J/cm2 caused a twofold decrease whereas UV-A did not affect glyphosate toxicity at doses ≤ 70 J/cm2. UV-C irradiation of glyphosate in drinking water and groundwater with naturally occurring organic and inorganic constituents showed comparable or greater reduction in toxicity compared to irradiation in deionized water. High-resolution mass spectrometry analyses of samples after UV-C irradiation showed > 90% decreases in glyphosate concentrations and the presence of multiple transformation products. The study suggests that UV mediated indirect photolysis can decrease concentrations of glyphosate and generate less toxic products with decreased overall toxicity to aquatic organisms.