The aim of this study was to investigate the effects of acute high-intensity interval training (HIIT) on immune function and oxidative stress in male canoe/kayak athletes who were well trained. A total of 22 participants were voluntarily recruited with an age range of 15.9 ± 2.3 years, height of 172.2 ± 5.5 cm, body mass of 63.30 ± 6.95 kg, and body fat of 13.77 ± 3.76%. The modified Wingate kayaking test on a kayak ergometer was performed by all participants. Blood samples were collected at three different time points: before the test (Pre-T), immediately after (Post-T), and 3 h post-test (Post-3 h). Saliva samples were collected at two different time points: before the test (Pre-T) and 3 h after the test (Post-3 h). Results indicated that acute canoe/kayak ergometry HIIT had significant effects on the percentages and counts of leukocytes, neutrophils, lymphocytes, and lymphocyte subsets. Additionally, it resulted in increased total LPS-stimulated neutrophil elastase release and alterations in plasma concentrations of superoxide dismutase, catalase, and TBARS. These findings suggest that conventional kayak HIIT regimens can have short-term effects on immune function and induce oxidative stress in athletes.