The aim of this study was to evaluate the effect of creatine supplementation on homocysteine (Hcy) metabolism after acute aerobic and anaerobic exercise. A total of 112 Wistar rats were divided into four groups: aerobic exercise (A), aerobic exercise plus creatine supplementation (ACr), anaerobic exercise (An), and anaerobic exercise plus creatine-supplemented (AnCr). Creatine supplementation consisted of the addition of 2% creatine monohydrate to the diet. After 28 days, the rats performed an acute moderate aerobic exercise bout (1 h swimming with 4% of total body weight load) or an acute intense anaerobic exercise bout (6 × 30-s vertical jumps into the water with a 30-s rest between jumps, with 50% of total body weight load). The animals were killed before (pre) and at 0, 2, and 6 h (n = 8) after acute exercise. Plasma Hcy concentration increased significantly (P < 0.05) up to 2 h after anaerobic exercise (An group: pre 8.7 ± 1.2, 0 h 13.2 ± 2.3, 2 h 13.5 ± 4.2, and 6 h 12.1 ± 2.2, μmol/l). The same did not occur in acute aerobic exercised animals. Nevertheless, creatine supplementation significant decreased (P < 0.05) homocysteine concentration independent of exercise intensity (AnCr group: pre 17%, 0 h 80%, 2 h 107%, and 6 h 48%; ACr group: pre 17%, 0 h 19%, 2 h 28%, and 6 h 27%). Increased S-adenosylhomocysteine was also found in the An group. In conclusion, acute intense anaerobic exercise increased plasma Hcy concentration. On the other hand, creatine supplementation decreased plasma Hcy independent of exercise intensity.