IMPORTANCE The tinnitus research literature suggests that N-methyl-D-aspartate (NMDA) receptor antagonists May be useful in reducing tinnitus. Nitrous oxide, a member of the NMDA receptor antagonist class, is a widely used general anesthetic and sedative with a proven safety record. OBJECTIVE To investigate whether nitrous oxide can reduce bothersome tinnitus. DESIGN, SETTING, AND PARTICIPANTS Randomized, placebo-controlled crossover trial conducted between October 15, 2016, and June 22, 2017. Participants attended 2 interventional sessions separated by at Least 14 days and were randomized to receive either placebo first or nitrous oxide first. Participants were followed up through completion of the second arm of the study. The setting was a clinical research unit at an academic medical center. Adults aged 18 to 65 years with subjective, idiopathic, nonpulsatile bothersome tinnitus of 6 months’ duration or longer were recruited from 2 clinical research databases. Seventy-one individuals were screened, of whom 40 were enrolled. Of those enrolled, 37 participants completed all components of the study. INTERVENTIONS The placebo session consisted of 50% nitrogen and 50% oxygen inhaled for 40 minutes, and the treatment session consisted of 50% nitrous oxide and 50% oxygen inhaled for 40 minutes. MAIN OUTCOMES AND MEASURES Tinnitus was assessed before and after intervention, with the change in the Tinnitus Functional Index (TFI) as the primary outcome measure. Secondary outcome measures included the Patients’ Global Impression of Change score and the change in the Global Bothersome Scale score. RESULTS Among 40 participants in this intent-to-treat randomized clinical trial with 20 participants randomly assigned to each group, the mean (SD) age of participants was 52.9 (11.1) years, with equal numbers of male and female participants. The TFI after intervention was a mean (SD) of 1.8 (8.8) points lower than before intervention in the placebo arm and a mean (SD) of 2.5 (11.0) points lower than before intervention in the nitrous oxide arm. The within-participant mean difference in the change in the TFI of the placebo arm compared with the nitrous oxide arm was −1.1 points (95% CI, −5.6 to 3.4 points). The difference between the placebo and nitrous oxide arms was neither clinically meaningful nor statistically significant. CONCLUSIONS AND RELEVANCE Nitrous oxide was no more effective than placebo for the treatment of subjective, idiopathic tinnitus.