The demand of glyphosate-based herbicides including Roundup® is rising in the tropics due to increase occurence of glyphosate-resistant weeds that require higher herbicide application rates but also because of their use associated with genetically engineered, glyphosate-tolerant crops. Consequently, there is now an excessive use of glyphosate in agricultural areas with potential adverse effects also for the surrounding aquatic environments. This study aimed to determine the sensitivity of the freshwater planarian Girardia tigrina to acute and chronic exposures of Roundup®. Planarians were exposed to a range of lethal and sub-lethal concentrations of Roundup® to determine the median lethal concentration (LC) concerning its active ingredient glyphosate and also effects on locomotor velocity (pLMV), feeding rate, regeneration, reproductive parameters and morphological abnormalities. Regeneration endpoints included length of blastema and time for photoreceptors and auricles regeneration after decapitation, while effects on reproduction were assessed measuring fecundity (number of deposited cocoons) and fertility (number of hatchlings) over five weeks of exposure to glyphosate. The estimated 48 h LC of was 35.94 mg glyphosate/L. Dose dependent effects were observed for feeding, locomotion and regeneration endpoints with Lowest observed effect concentration (LOEC) values as low as 3.75 mg glyphosate/L. Chronic exposures to environmentally relevant concentrations of glyphosate significantly impaired fecundity and fertility rates of exposed planarians (median effective concentration, EC = 1.6 mg glyphosate/L for fecundity and fertility rates). Our results show deleterious effects of Roundup® on regeneration, behavior and reproduction of freshwater planarians and add important ecotoxicological data towards the environmental risk assessment of glyphosate-based herbicide in freshwater ecosystems.