Previous studies on caffeine-induced effects on endurance and performance have been on skeletal muscle and brain metabolism but not on cardiac tissue metabolism. This study investigated the effect of caffeine on cardiac tissue substrate metabolism in the rabbit. The study was carried out on adult male New Zealand rabbits divided into 3 groups (n=5). Group I rabbits served as control and were given distilled water orally while groups II and III rabbits were administered orally with 2mg/Kg and 6mg/kg of caffeine respectively for 28 days. Blood samples were collected by retro orbital puncture for biochemical analyses of serum myeloperoxidase activity, nitrites, total cholesterol, triglycerides and glucose concentrations. Animals were sacrificed by cervical dislocation and cardiac tissue biopsies were collected for biochemical and immunohistochemical analyses. Cardiac tissue glycogen concentration was determined by anthrone reagent method. Cardiac tissue CPT1 activity and cAMP concentration were determined by immunohistochemistry and colorimetry techniques respectively, with assay kits obtained from Biovision Inc. Values are means ± SEM, compared by ANOVA and Tukey Post Hoc test for honestly significant difference at α0.05. The results showed that Caffeine (2mg/kg and 6mg/kg) significantly reduced MPO activity from 0.72±0.05 to 0.164±0.045 and 0.46±0.12 U/L respectively and significantly increased serum nitric oxide only at 6mg/Kg, from 28.01±6.53 to 45.25±3.88µM of nitrite. Caffeine also increased serum total cholesterol and triglycerides at the two doses but the serum glucose increased significantly only at 6 mg/kg of caffeine. Also, both doses of caffeine increased cardiac tissue glycogen from 15.62±0.73 to 40.69±6.35 and 38.82±6.91mg/100g respectively while carnitine palmytol transferase 1 activity increased from 18.3% to 20% and 25.2% respectively. The study showed that caffeine exhibits strong effects on cardiac tissue metabolism most probably by causing cardio protection, cardiac hyperaemia, glycogen sparing, increased cAMP concentration and cardiac utilization of fatty acids as source of energy.