Glyphosate-based herbicides (GBH) are one of the most used herbicide nowadays, whilst there is growing concern over their impact on aquatic environment. Since data about the early proteomic response and toxic mechanisms of GBH in fish is very limited, the aim of this study was to investigate the early toxicity of GBH in the gills of guppies Poecilia reticulata using a proteomic approach associated with histopathological index. Median lethal concentration (LC50,96 h) was determined and LC50,96h values of guppies exposed to GBH were 3.6 ± 0.4 mg GLIL(-1). Using two-dimensional gel electrophoresis associated with mass spectrometry, 14 proteins regulated by GBH were identified, which are involved in different cell processes, as energy metabolism, regulation and maintenance of cytoskeleton, nucleic acid metabolism and stress response. Guppies exposed to GBH at 1.82 mg GLIL(-1) showed time-dependent histopathological response in different epithelial and muscle cell types. The histopathological indexes indicate that GBH cause regressive, vascular and progressive disorders in the gills of guppies. This study helped to unravel the molecular and tissue mechanisms associated with GBH toxicity, which are potential biomarkers for biomonitoring water pollution by herbicides.