Graphene oxide (GO) is a very attractive material for use in a vast number of applications. However, before its widespread use, it is important to consider potential issues related to environmental safety to support its safe application. The aim of this study was to investigate effects on fish (rainbow trout) following GO exposure. Using both an in vitro approach with the RTL W1 rainbow trout liver cell line, and in vivo exposures, following OECD TG 203, disturbances at the cellular level as well as in the gills and liver tissue of juvenile trout were assessed. In RTL W1 cells, a time and concentration-dependent loss in cell viability, specifically plasma membrane integrity and lysosomal function, was observed after 96 hours of exposure to GO at concentrations ≥ 18.75 mg/L. Additionally, increased reactive oxygen species (ROS) levels were evidenced at concentrations ≥ 18.75 mg/L, and an enhancement of metabolic activity was noted with concentrations ≥ 4.68 mg/L. In vivo exposures to GO did not provoke mortality in rainbow trout juveniles following 96 h exposure but led to histological alterations in gills and liver tissues, induction of enzymatic detoxification activities in the liver, as well as aryl hydrocarbon receptor (ahr)-cytochrome P450 1a (cyp1a) gene expression downregulation, and upregulation of pro-inflammatory cytokines il1b and il8 at GO concentrations ≥ 9.89 mg/L.