PPARα plays a central role in the regulation of cellular lipid metabolism in tissues with high capacity for fatty acid oxidation, such as heart, brown adipose tissue, slow-twitch skeletal muscle, and liver. We postulated that PPARα plays is pivotal to cardiac metabolic response to chronic hypoxia and used high fat (HF) feeding and gene deletion to alter PPARα expression. Control mice (129EvSv) and PPARα-/- mice were exposed to 3 weeks of normobaric hypoxia at 11% oxygen (n=70), or to normoxia (n=84). To stimulate the PPARα pathway, half the mice were fed a HF diet (55% fat), and controls were chow-fed (7.5% fat). Mice were anesthetised with 1.5% isoflurane in O2 and in vivo cardiac function was measured using cine MRI. Hearts were isolated and perfused to measure palmitate oxidation and glycolytic flux using 3H labelling. Cardiac function in control mice was unaffected by chronic hypoxia, but palmitate oxidation was reduced by 23% (p<0.05) and glycolytic flux increased 2-fold (p<0.01). PPARα mRNA expression was decreased by 31% (p<0.05) in hypoxic chow fed mice, which reduced protein levels of UCP3 by 37% (p<0.05) and MTE-1 by 39% (p<0.01). HF feeding during hypoxia decreased cardiac output by 24% (p<0.01), decreased ejection fraction by 9% (p<0.05), and increased palmitate oxidation by 30% (p<0.01) compared with normoxic controls. HF feeding increased PPARα mRNA expression 4-fold (p<0.05) and doubled UCP3 and MTE-1 (p<0.05) levels, and were unchanged by hypoxia. PPARγ and PPARβ/δ protein levels were unaffected by hypoxia or HF feeding. Hypoxic PPARα-/- hearts had 19% (p<0.01) lower cardiac output than normoxic controls, with cardiac metabolism and UCP3 level unaltered by hypoxia and/or HF feeding. In conclusion, feeding a high fat diet under hypoxic conditions prevented metabolic adaptation to hypoxia, with cardiac substrate metabolism similar to that of normoxic high fat fed mice. With scarce oxygen supply, the increase in UCP3 may have decreased the efficiency of mitochondrial ATP synthesis and impaired cardiac function following chronic hypoxia. Deletion of PPARα also impaired cardiac function as metabolic adaptation was prevented. Therefore, cardiac metabolic adaptation via PPARα is essential to maintain cardiac function following chronic hypoxia.