Acclimation to abiotic challenges, including decreases in O2 availability, requires physiological and anatomical phenotyping to accommodate the organism to the environmental conditions. The retention of a nucleus and functional mitochondria in mature fish red blood cells makes blood a promising tissue to analyse the transcriptome and metabolic responses of hypoxia-challenged fish in an integrative and non-invasive manner.Juvenile gilthead sea bream (Sparus aurata) were reared at 20-21 °C under normoxic conditions (> 85% O2 saturation) followed by exposure to a gradual decrease in water O2 concentration to 3.0 ppm (41-42% O2 saturation) for 24 h or 1.3 ppm (18-19% O2 saturation) for up to 4 h. Blood samples were collected at three different sampling points for haematological, biochemical and transcriptomic analysis.Blood physiological hallmarks remained almost unaltered at 3.0 ppm, but the haematocrit and circulating levels of haemoglobin, glucose and lactate were consistently increased when fish were maintained below the limiting oxygen saturation at 1.3 ppm. These findings were concurrent with an increase in total plasma antioxidant activity and plasma cortisol levels, whereas the opposite trend was observed for growth-promoting factors, such as insulin-like growth factor I. Additionally, gene expression profiling of whole blood cells revealed changes in upstream master regulators of mitochondria (pgcβ and nrf1), antioxidant enzymes (gpx1, gst3, and sod2), outer and inner membrane translocases (tom70, tom22, tim44, tim10, and tim9), components of the mitochondrial dynamics system (mfn2, miffb, miro1a, and miro2), apoptotic factors (aifm1), uncoupling proteins (ucp2) and oxidative enzymes of fatty acid β-oxidation (acca2, ech, and hadh), the tricarboxylic acid cycle (cs) and the oxidative phosphorylation pathway. The overall response is an extensive reduction in gene expression of almost all respiratory chain enzyme subunits of the five complexes, although mitochondrial-encoded catalytic subunits and nuclear-encoded regulatory subunits of Complex IV were primarily increased in hypoxic fish.Our results demonstrate the re-adjustment of mitochondrial machinery at transcriptional level to cope with a decreased basal metabolic rate, consistent with a low risk of oxidative stress, diminished aerobic ATP production and higher O2-carrying capacity. Taken together, these results suggest that whole blood cells can be used as a highly informative target tissue of metabolic condition.