Purpose: This study aimed to determine the effects of heat acclimatization on performance and pacing during outdoor cycling time trials (TT, 43.4 km) in the heat. Methods: Nine cyclists performed three TT in hot ambient conditions (TTH, approximately 37-C) on the first (TTH-1), sixth (TTH-2), and 14th (TTH-3) days of training in the heat. Data were compared with the average of two TT in cool condition (approximately 8-C) performed before and after heat acclimatization (TTC). Results: TTH-1 (77 T 6 min) was slower (P = 0.001) than TTH-2 (69 T 5 min), and both were slower (P G 0.01) than TTC and TTH-3 (66 T 3 and 66 T 4 min, respectively), without differences between TTC and TTH-3 (P 9 0.05). The cyclists initiated the first 20% of all TT at a similar power output, irrespective of climate and acclimatization status; however, during TTH-1, they subsequently had a marked decrease in power output, which was partly attenuated after 6 d of acclimatization and was further reduced after 14 d. HR was higher during the first 20% of TTH-1 than that in the other TT (P G 0.05), but there were no differences between conditions from 30% onward. Final rectal temperature was similar in all TTH (40.2-C T 0.4-C, P = 1.000) and higher than that in TTC (38.5-C T 0.6-C, P G 0.001). Conclusions: After 2 wk of acclimatization, trained cyclists are capable of completing a prolonged TT in a similar time in the heat compared with cool conditions, whereas in the unacclimatized state, they experienced a marked decrease in power output during the TTH.