Betaine (BET) has shown to be effective in improving body composition and performance, although research in women is lacking. This study investigated the effects of BET supplementation on markers of metabolic flexibility, body composition, and anaerobic performance in college females. Twenty-three active subjects with 21.8 ± 3.0 years of age, 66.6 ± 8.8 kg body mass, 1.6 ± 0.1 m height, and 23.2 ± 5.3% body fat performed a graded exercise test on a cycle ergometer consisting of 4 incremental, 3 min stages for collection of fat and carbohydrate oxidation rates. Three 10 s sprint tests were then completed against a resistance of 7.5% of body mass, separated by 2.5 min of recovery. The study comprised 3 phases: (a) pre-supplementation, (b) randomization to supplement for 2-weeks with either 2.4 g/day BET or placebo (parallel design), and (c) post-supplementation. Repeated-measures analysis of variance were conducted to determine interactions or main effects. There were no group differences for substrate oxidation rates (p > 0.05). Although body composition improved pre-post for both groups (p < 0.05), only the BET group experienced a significant increase in fat free mass (p < 0.01; ∼3%). Further, only the BET group experienced improvements to performance such as a higher mean power output during the final sprint (p = 0.02; ∼3%) and a lower RPE during the final stage of the graded exercise test (p = 0.02). Results from this study suggest BET supplementation may improve body composition and some markers of performance during exercise in collegiate women.