Exposure to oil-derived contaminants directly affect the transcriptional regulation of the cells. In the present study we evaluate the impact of the petroleum-water accommodated fraction (WAF) from a light crude oil on the transcriptome wide regulation of gills and liver of Achirus lineatus (Linnaeus, 1785), a flounder native from the Gulf of Mexico (GM). Adults individuals of A. lineatus were experimentally exposed to a sublethal WAF/water mix of 50% v/v during 48 h. These results indicate that exposure to WAF produced a more dramatic transcriptional changes in gill than in liver. We also identified the regulation of xenobiotic, and redox metabolism, and the turn on of DNA repair mechanisms. Importantly, we observed an important number of hypoxia-related genes, including the hypoxia-inducible factor-1 alpha, as well as multiple signaling pathways. The metabolic consequences of these regulatory mechanisms provide of a deeper understanding of the consequences to a sub-lethal and acute exposure to WAF. Data generated provide of new insights into the mechanisms of WAF-induced toxicity in flounders, and could be also a very important for the development of crude oil biomonitoring strategies using this species.