Research has shown that chronic, long and exhausting endurance training programs cause iron deficiency. This problem can have a negative effect on physical performance. The aim of this research was to study the effect of endurance training and iron supplement on anemic indexes [hemoglobin (Hb), hematocrit (HCT), red blood cell (RBC), total iron binding capacity (TCBC) and ferritin], and cytochrome C oxidase (COX) enzyme in male rats. Forty male wistar 4848 rats with an average weight of 321.55+22.67 were divided into four groups: experimental group I, experimental group II, control I, and control II. Experimental group I were trained to perform endurance running on a treadmill for 12 weeks (with an intensity of 32 m.min-1 for 60 minutes every session, five sessions a week) (T). Experimental group II received 800 micrograms of iron supplement (ferrous sulfate) daily (gavage) in addition to being trained to perform endurance running on the treadmill (Ti). Control group I were sedentary (inactive) for 12 weeks (S) and control group II did not perform any exercise but received iron supplement as did experimental group II (Si). After 12 weeks, rats were killed by the easy method. Blood samples were analyzed for anemia indexes in the laboratory. Muscle soleus COX was measured by cytochrome C oxidase assay kit. The data were analyzed by one-way ANOVA with (P<0.05). Statistical analyses showed a disorder in the capacity of iron resources in the sample. On the other hand, the consumption of iron supplement not only improved anemia indexes but also increased the activity of COX in Ti group. But iron supplement had no positive effect on inactive (sedentary) sample. The most important conclusion is that iron supplement is very useful and effective for subjects with iron deficiency.