Roundup(®) (glyphosate formulation) is a nonselective and posts emergent herbicide used for controlling aquatic weeds and different concentrations are used in cultures around the world. The objective of this investigation was to examine the effects of Roundup(®) (glyphosate formulation) on the biochemical composition, levels of lipoperoxidation, Na(+)/K(+)ATPase activity and reproductive traits in the Hyalella castroi. Amphipods were collected in summer 2009, in the southern Brazilian highlands. In the laboratory, the animals were kept in aquariums under controlled conditions for 7 days, and after this period they were exposed to 0.36, 0.52, 1.08 and 2.16 mg/l of glyphosate for 7 days. After the period of exposure, the animals were immediately frozen for determination of glycogen, proteins, lipids, triglycerides, cholesterol, levels of lipoperoxidation, and Na(+)/K(+)ATPase activity. During each day of the cultivation reproductive traits (number of reproductive pairs, ovigerous females and eggs in the marsupium) were observed. All concentrations of Roundup(®) induced significant decreases in all biochemical parameters and Na(+)/K(+)ATPase activity, and significant increase in lipoperoxidation levels. Showing this form a potentially toxic effect at very low concentrations, this pattern of results can lead to significant changes in trophic structure of limnic environments because these amphipods are important links in food chain in these habitats.