Summary Nitrous oxide inactivates vitamin B12 with detrimental consequences for folate and methionine metabolism. This inactivation can be clinically detected by an increase in plasma total homocysteine. In a clinical trial, we tested the hypothesis that a preoperative infusion of vitamin B12 and folate prevents the nitrous oxide-induced homocysteine increase. Sixty-three healthy patients scheduled for elective surgery were randomly allocated to receive (a) B-vitamins and nitrous oxide; (b) placebo and nitrous oxide or (c) normal saline and no nitrous oxide. Fifty-nine patients were included in the final study population. After intravenous B-vitamin infusion, plasma vitamin B12 and folate concentrations increased 35-fold and 12-fold on the first postoperative measurement, respectively. Patients who received B-vitamins developed a similar increase (+18%) in homocysteine after nitrous oxide (+ 1.9 μmol.L−1; 95% CI: 0.2 – 3.6 μmol.L−1) as patients who did not receive B-vitamins (+ 22%; + 2.7 μmol/L; 95% CI: 0.6 – 4.8 μmol.L−1). Patients who did not receive nitrous oxide (“air control”) had no change in homocysteine (+ 0.5 μmol.L−1; 95% CI: −0.8 – 1.9 μmol.L−1). This trial indicates that preoperative IV B-vitamins may not prevent nitrous oxide-induced hyperhomocysteinemia.