Carbohydrate (CHO) supplementation and female sex independently influence the natural killer (NK) cell response to acute exercise. Consequently, this study sought to elucidate sex-based differences in the distribution of NK cell subsets (i.e., CD56dimand CD56bright) in response to exercise and CHO intake. Twenty-two healthy 14-yr-old girls ( n = 11) and boys ( n = 11) cycled for 60 min at 70% maximal oxygen consumption while drinking 6% CHO (CT) or flavored water (WT). Blood was collected at rest, during exercise (30 and 60 min), and during recovery (30 and 60 min) to identify CD3−CD56dimand CD3−CD56brightNK cells. The activation marker CD69 was also determined on CD3−CD56+cells. CD56dimresponses, expressed as proportions or cell counts, were greater ( P ≤ 0.01) in girls by 67 and 105%, respectively. CD56brightcell counts ( P = 0.006), but not CD56brightproportions ( P = 0.89), were greater in girls by 82%. Both CD56dimand CD56brightsubset responses, expressed as proportions or cell counts, were lower ( P ≤ 0.01) in CT vs. WT by 33–36%. The CD56bright-to-CD56dimratio decreased at 30 min of exercise but increased during recovery ( P < 0.001), with no effect of sex or CHO. Regardless of trial, CD3−CD56+cells expressed ∼18% higher levels of CD69 during recovery in girls but not boys ( P = 0.03), despite similar proportions and counts of CD69+cells. These results demonstrate sex-based differences in the distribution of NK cell subsets and activation status in response to exercise, but not CHO intake, and further support the need to control for sex in exercise immunology studies.