Dietary nitrate (NO 3− ) supplementation via beetroot juice (BR) is known to improve endurance performance in untrained and moderately trained individuals. However, conflicting results exist in well-trained individuals. Evidence suggests that the effects of NO 3− are augmented during conditions of reduced oxygen availability (e.g., hypoxia), thereby increasing the probability of performance improvements for well-trained athletes in hypoxia vs. normoxia. This randomized, double-blinded, counterbalanced-crossover study examined the effects of 7 days of BR supplementation with 12.4 mmol NO 3− per day on 10-km cycling time trial (TT) performance in 12 well-trained cyclists in normoxia (N) and normobaric hypoxia (H). Linear mixed models for repeated measures revealed increases in plasma NO 3− and NO 2− after supplementation with BR (both p < 0.001). Further, TT performance increased with BR supplementation (∼1.6%, p < 0.05), with no difference between normoxia and hypoxia (p = 0.92). For respiratory variables there were significant effects of supplementation on VO 2 (p < 0.05) and VE (p < 0.05) such that average VO 2 and VE during the TT increased with BR, with no difference between normoxia and hypoxia (p ≥ 0.86). We found no effect of supplementation on heart rate, oxygen saturation or muscle oxygenation during the TT. Our results provide new evidence that chronic high-dose NO 3− supplementation improves cycling performance of well-trained cyclists in both normoxia and hypoxia.