Dietary nitrate (NO3-) 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 NO3- 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 NO3- 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 NO3- and NO2- 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 VO2 (p < 0.05) and VE (p < 0.05) such that average VO2 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 NO3- supplementation improves cycling performance of well-trained cyclists in both normoxia and hypoxia.