This investigation examined the effect of beetroot juice (BR) supplementation, a source of dietary nitrate (NO3 −), on cycling time-trial (TT) performance and thermoregulation in the heat. In a double-blind, repeated-measures design, 12 male cyclists (age 26.6 ± 4.4 years, VO2peak 65.8 ± 5.5 mL.kg−1.min−1) completed four cycling TTs (14 kJ.kg−1) in hot (35°C, 48% relative humidity) and euthermic (21°C, 52%) conditions, following 3 days supplementation with BR (6.5 mmol NO3 − for 2 days and 13 mmol NO3 − on the final day), or NO3 –depleted placebo (PLA). Salivary NO3 − and nitrite, core (Tc) and mean skin temperature (Tsk) were measured. Salivary NO3 − and nitrite increased significantly post-BR supplementation (p < 0.001). Average TT completion time (mm:ss) in hot conditions was 56:50 ± 05:08 with BR, compared with 58:30 ± 04:48 with PLA (p = 0.178). In euthermic conditions, average completion time was 53:09 ± 04:35 with BR, compared with 54:01 ± 04:05 with PLA (p = 0.380). The TT performance decreased (p < 0.001), and Tc (p < 0.001) and Tsk (p < 0.001) were higher in hot compared with euthermic conditions. In summary, BR supplementation has no significant effect on cycling TT performance in the heat.