Objective: Nitrous oxide is a common inhalation anesthetic agent in general anesthesia. While it is widely accepted as a safe anesthetic agent, evidence suggests exposure to this gas, leads to hyperhomocysteinemia. The present study aimed to evaluate the effects of single-dose intravenous infusions of vitamin B12, before and after the induction of nitrous oxide anesthesia on homocysteine levels after the surgery. Methods: This double-blind randomized controlled trial was conducted on 60 patients who were scheduled for elective surgery under general anesthesia, presumably lasting for more than two hours. The subjects were randomly allocated to three groups of 20. For the first group, vitamin B12 solution (1 mg/100 ml normal saline) and 100 ml of normal saline (placebo), were infused before and after the induction of anesthesia, respectively. The second group received placebo and vitamin B12 infusion before and after the induction of anesthesia, respectively. The third group received placebo infusions at both times. Homocysteine levels were measured before and 24 hours after the surgery. Results: The mean homocysteine and vitamin B12 levels were significantly different within the three groups (p<0.001). In patients who had been infused with vitamin B12 before the surgery, homocysteine levels were significantly lower than the other two groups. In the placebo group, homocysteine levels significantly increased after the surgery. Conclusion: Nitrous oxide causes hyperhomocysteinemia after general anesthesia. Since vitamin B12 infusion is a safe and inexpensive method to decrease homocysteine levels in these patients, it may be recommended for patients undergoing nitrous oxide anesthesia to be used before induction of anesthesia.