The objective of this study was to determine the effects of supplementation of ascorbic acid, Vitamin E (Vit. E) and their combination in drinking water on sperm characteristics, lipid peroxidation (LPO) and seminal plasma enzymes of mature male rabbits. Twenty-four male New Zealand White rabbits (5 months old) were given drinking water supplemented with ascorbic acid (1.5 g/l), Vit. E (1.0 g/l) and ascorbic acid+Vit. E (1.5+1.0 g/l) for 12 weeks. Vitamin supplementation in drinking water increased feed intake, but body weight gain was not significantly affected. Concentrations of thiobarbituric acid-reactive substances (TBARS) were significantly (P<0.05) reduced in seminal plasma of treated groups compared with the control. Treatment with ascorbic acid, Vit. E, and their combination significantly (P<0.05) increased lipido (reaction time), ejaculate volume, sperm concentration, total sperm output, sperm motility index, total motile sperm, packed sperm volume, initial hydrogen ion concentration (pH), and semen initial fructose concentration. Abnormal and dead sperm were significantly (P<0.05) decreased in treated animals. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were significantly (P<0.05) decreased, whereas glutathione S-transferase (GST) showed a significant increase in seminal plasma of treated animals compared with the controls. The results from this study indicated that supplementation of drinking water with antioxidant ascorbic acid, Vit. E and their combination reduced the production of free radicals and can improve rabbit semen quality, but the greater improvement seemed to be from Vit. E.