Hypoxia is a common concern in shrimp aquaculture, affecting growth and survival. Although recent studies have revealed important insights into hypoxia in shrimp and crustaceans, knowledge gaps remain regarding this stressor at the molecular level. In the present study, a gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach was employed to characterize the metabolic pathways underlying responses of shrimp (Penaeus vannamei) to hypoxia and to identify candidate biomarkers. We compared metabolite profiles of shrimp haemolymph before (0 h) and after exposure to hypoxia (1 & 2 h). Dissolved oxygen levels were maintained above 85% saturation in the control and before hypoxia, and 15% saturation in the hypoxic stress treatment. Results showed 44 metabolites in shrimp haemolymph that were significantly different between before and after hypoxia exposure. These metabolites were energy-related metabolites (e.g., TCA cycle intermediates, lactic acids, alanine), fatty acids and amino acids. Pathway analysis revealed 17 pathways that were significantly affected by hypoxia. The changes in metabolites and pathways indicate a shift from aerobic to anaerobic metabolism, disturbance in amino acid metabolism, osmoregulation, oxidative damage and Warburg effect-like response caused by hypoxic stress. Among the altered metabolites, lactic acid was most different between before and after hypoxia exposure. Biomarker analysis also indicated lactic acid as biomarker for hypoxia and model prediction. Future investigations may validate this molecule as a stress biomarker in aquaculture. This study contributes to better understanding of hypoxia in shrimp and crustaceans at the metabolic level and provides a base for future metabolomics investigations on hypoxia.