Alkaline water have been claimed to boost antioxidant mechanisms. Thus, this study aimed to investigate if Zamzam water that is similarly alkaline, can promote antioxidant mechanisms in normal rats and those stressed with the high dose of gentamicin. In one experiment, the effects of Zamzam water were compared to those of ordinary bottled water in normal rats. In the second experiment, the effects of Zamzam water were compared to those of ordinary bottled water in rats injected intraperitoneally with either saline or gentamicin at a dose of 80 mg/kg body weight. No significant differences in serum levels of a group of trace elements including arsenic were observed between the groups of rats given the two types of water. Serum antioxidant enzymes (catalase and dismutase) were similar between the different groups of rats both in normal or those stressed by gentamicin injection. In the two groups injected with gentamicin, total antioxidant capacity was significantly higher in the group given Zamzam water. In conclusion, giving Zamzam water to normal rats for three weeks does not show any sign of toxicity and seems to be associated with increased total antioxidant in rats stressed with gentamicin overdose. Further studies are needed to evaluate these findings.