Hypoxia-inducible factor (HIF) is a transcription factor which plays a pivotal role in the cellular response to reduced oxygen availability. Manipulation of the HIF system could have therapeutic potential in the treatment of ischaemic cardiac disease. HIF activity is regulated by two families of oxygen sensitive enzymes; the prolyl hydroxylase domain (PHD) family, and factor-inhibiting HIF (FIH1). The role of FIH1 in the heart is unknown. We compared cardiac function and metabolism in hearts from mice with a null mutation in the FIH1 gene (FIH1-/-, n≥5 hearts) and wild type littermate controls (WT, n≥6 hearts). In vivo cardiac function was investigated using cine MRI in anaesthetized mice (2% isoflurane maintenance). Individual ventricular myocytes were isolated by collagenase digestion and contractility measured using an inverted fluorescence microscope (IonOptix sarcomere length detection, 32°C, 1 Hz stimulation, n≥50 cells per group). Intracellular calcium (Ca2+) transients were recorded using the fluorescent Ca2+ probe, fura-2. Glycolytic flux was investigated in Langendorff perfused beating hearts using 3H labelling techniques. Data are expressed as mean ± standard error and compared using unpaired t-tests. In vivo cardiac function was impaired in FIH1-/- mice, with stroke volume (23.4 ± 1.5 µl) reduced by 15% in FIH1-/- hearts compared to WT (27.4 ± 2.3 µl, p<0.05). Contractility was reduced in myocytes isolated from FIH1-/- hearts, with percentage sarcomere shortening significantly lower in FIH1-/- cells (3.01 ±0.20 %) than in WT cells (3.92 ± 0.17 %). This was accompanied by reduced Ca2+ transient amplitude (fura-2 ratio 0.21 ± 0.02 in FIH1-/-, compared to 0.29 ± 0.02 in WT, p<0.05). Furthermore, the time from peak Ca2+ transient to 50% decline (RT50) was significantly slower in FIH1-/- (198 ± 8 ms) than WT cells (150 ± 6 ms, p<0.05). Reduced Ca2+ transient amplitude and impaired Ca2+ transient decline in FIH1-/- myocytes persisted during β-adrenergic stimulation (10 nM isoprenaline). Hence, Ca2+ transient amplitude and RT50 were 0.39 ± 0.03 and 96 ± 5 ms respectively in FIH1-/- compared to 0.48 ± 0.02 and 78 ± 3 ms respectively in WT (p<0.05). Cardiac metabolism was also altered in FIH1-/- mice. Glycolytic flux was significantly higher in FIH1-/- hearts (1.17 ± 0.04 µmol/min/g) than WT (0.79 ± 0.12 µmol/min/g, p<0.05). Furthermore, FIH1-/- hearts demonstrated increased pyruvate kinase and hexokinase activity. In conclusion, our data suggest a novel role for FIH1 in modulating cardiac E-C coupling and metabolism. Genetic ablation of FIH1 produced cardiac effects comparable to those observed following chronic hypoxic exposure, notably decreased contractility, impaired relaxation and increased glycolysis. Potential mechanisms for these observed changes are currently being explored.