AbstractDecades after being banned in many countries, DDT and its metabolites are still considered major environmental hazards. The p,p′‐DDE isomer, the DDT metabolite found in highest concentration in aquaculture feeds, is an endocrine disruptor with demonstrated ability to induce epigenetic effects. This study aimed at examining the impact of p,p′‐DDE on Atlantic salmon. Primary hepatocytes were exposed to four concentrations of p,p′‐DDE (0.1, 1, 10, 100 μm) for 48 hours, and endpoints included cytotoxicity, global DNA methylation, targeted transcription and metabolomics profiling (100 μm). p,p′‐DDE was moderately cytotoxic at 100 μm. No impact was seen on global DNA methylation. Vtg1 and esr1 transcription, markers of endocrine disruption, was most strongly induced at 10 μm p,p′‐DDE, while ar showed strongest response at 100 μm. Metabolomics profiling showed that p,p′‐DDE at 100 μm most strongly affected carbohydrate metabolism, primary bile acid metabolism, leucine, isoleucine and valine metabolism, diacylglycerol and sphingolipid metabolism. Observed changes in lipid levels suggest that p,p′‐DDE interferes with phospholipid membrane biosynthesis. Elevation of bile acid levels in p,p′‐DDE‐exposed hepatocytes indicates upregulation of synthesis of bile acids after cytochrome P450 activation. Pathway analysis showed that the superpathway of methionine degradation was the most significantly affected pathway by p,p′‐DDE exposure, while endocrine system disorder topped the diseases and disorder ranking. In conclusion, this work predicts an endocrine response to p,p′‐DDE exposure, and demonstrates how this legacy pesticide might interfere with mechanisms linked to DNA methylation in Atlantic salmon hepatocytes.