The beneficial effects of beetroot juice supplementation (BJS) have been tested during cycling, walking, and running. The purpose of the present study was to investigate whether BJS can also improve performance in swimmers. Fourteen moderately trained male master swimmers were recruited and underwent two incremental swimming tests randomly assigned in a pool during which workload, oxygen uptake (VO2), carbon dioxide production (VCO2), pulmonary ventilation (VE), and aerobic energy cost (AEC) of swimming were measured. One was a control swimming test (CSW) and the other a swimming test after six days of BJS (0.5l/day organic beetroot juice containing about 5.5 mmol of NO3−). Results show that workload at anaerobic threshold was significantly increased by BJS as compared to the CSW test (6.3 ± 1 and 6.7 ± 1.1 kg during the CSW and the BJS test respectively). Moreover, AEC was significantly reduced during the BJS test (1.9 ± 0.5 during the SW test vs. 1.7 ± 0.3 kcal·kg−1·h−1 during the BJS test). The other variables lacked a statistically significant effect with BJS. The present investigation provides evidence that BJS positively affects performance of swimmers as it reduces the AEC and increases the workload at anaerobic threshold.