The silverside (Odontesthes humensis) is a very interesting model for toxicological studies due its high sensitivity and need for good water quality. The aim of this study was to evaluate the effects of Roundup on spermatozoa of O. humensis, after acute exposure. The fish were exposed to 0 and 7.8 mg L-1 (a.e.) of glyphosate, respectively. Through computer-assisted sperm analysis, a significant decrease in concentration, total and progressive motility, average path distance, straight line distance, path average velocity, curved line velocity, straight line velocity linearity, wobble, amplitude of lateral head displacement, cross beat frequency, and motility period of silverside spermatozoa exposed to Roundup was observed. Also, increase in membrane fluidity, ROS production and lipid peroxidation and a decrease in the mitochondrial functionality was observed in spermatozoa of Roundup exposed silversides. It was demonstrated that Roundup exposure in a concentration that can be achieve in natural water bodies soon after its application in fields is able to cause losses in several sperm quality parameters, consequently decreasing the fertilization potential of O. humensis spermatozoa.