Roundup® (RD) is a glyphosate-based herbicide used to control weeds in agriculture, and fishponds. In the Amazon, hypoxia is a natural phenomenon in flooded areas. Beyond the challenge of hypoxia, fish need to cope with the use of pesticides as RD that increases in the aquatic environment through the leaching of agricultural areas, and in aquaculture fish tanks. Thus, there is a need to better understand the combined effects of hypoxia and RD contamination for aquatic biota. The aim of this study was to investigate the effects of Roundup® (RD) and subsequent acute hypoxia in the gene expression, genotoxicity, histological and physiological responses of Colossoma macropomum. Fish were individually exposed to four different treatments during 96 h: normoxia (N), hypoxia (H), RD plus normoxia (NRD), and RD plus hypoxia (HRD) (RD concentration represents 75% of LC - nominal concentration 15 mg L to C. macropomum). HRD fishes presented down-regulation of hif-1α gene and ras oncogene, while NRD fish presented overexpression of ras; no difference occurred in hif-1α gene expression in both normoxia treatments. The glutathione-S-transferase and catalase activities increased in HRD fish liver compared to NRD. Otherwise, there was no difference in lipoperoxidation (LPO) between all treatments. Genetic Damage Index, measured throughout comet assay in erythrocytes of all treatments, presented similar values, excepted by fish exposed to NRD. As regard as hypoxic exposure, hypoxic fish presented significantly lower values, compared to HRD fishes. An increase in liver histological injuries occurred in H and HRD fish groups. In conclusion, we may affirm that C. macropomum is sensitive concerning RD contamination and that this sensitivity increases when combined with hypoxia.