Vitamin C consumption is important in Iron absorption. Therefore, the purpose of this study was to investigate the effect of acute aerobic activity after two weeks vitamin C supplementation on Iron status and hematological indices in club football players. In this quasi-experimental study, 40 club football players from Khuzestan province were randomly divided into two groups of vitamin C and placebo. Daily and for 14 days, subjects received 400 mg vitamin C in vitamin C group and 400mg dextrose in placebo group. 48 hours after the end of supplementation, a Bruce test was performed. To measure Iron, Ferritin, RBC and Hb levels, four blood samples were taken from the subjects, before and after the supplementation, immediately after and 72 hours after the Bruce test. Repeated measure ANOVA and Two way analysis of variance was used. 2 weeks of vitamin C supplementation showed a significant increase in RBC (p=0.026), Hb (p=0.041), serum Iron (p=0.008) and serum Ferritin (p=0.004) compared to placebo group. After aerobic exhaustive activity, RBC (p=0.034), serum Iron (p=0.023) and Ferritin (p=0.031) in vitamin C group significantly decreases compared to placebo group. After 72 hours of recovery, the vitamin‌ C group showed a significant increase in serum Iron (p=0.036) and Ferritin (p=0.045) compared to placebo group. It seems that two weeks of vitamin C supplementation can improve the serum Iron, Ferritin, RBC and Hb profile of professional footballers before aerobic exhaustive activity and in recovery.