Importance: One-third of singers and vocal professionals report experiencing a benefit from empirical vitamin B12injections for improvement of mild singing-related symptoms (eg, reduced stamina, vocal fatigue, and effort). However, there is no objective evidence to support or refute these claims. Objective: To assess the presence and magnitude of the effect of empirical vitamin B12injection on the vocal performance of singers. Design, Setting, and Participants: A randomized, double-blind, placebo-controlled, crossover trial was conducted from November 7, 2017, to November 30, 2018, at an academic voice center among 20 active adult singers without dysphonia but with mild vocal symptoms. Individuals with known or suspected vitamin B12deficiency or active or recent vitamin B12treatment were excluded. Analysis was on a per-protocol basis. Interventions: Participants were randomized to receive an intramuscular (deltoid) injection of either vitamin B12(1000 μg of cyanocobalmin) or placebo (0.9% sodium chloride). After a washout period of at least 4 weeks, participants were crossed over to receive the opposite injection. Both the investigators and participants were blinded to the order of injections. Main Outcomes and Measures: The participants completed the Singing Voice Handicap Index-10 (SVHI-10), the Voice Fatigue Index (VFI), and the Evaluation of the Ability to Sing Easily (EASE) before each injection and at intervals of 1 hour, 3 hours, 24 hours, 72 hours, and 1 week after the injection. The primary time point assessment was 72 hours after injection, and the SVHI-10 score was the primary outcome measure. Results: Twenty singers (10 men; median age, 22 years [range, 19-42 years]) were enrolled. The improvements after either placebo or vitamin B12injections were comparable to each other. At 72 hours after the vitamin B12injection, the median difference in the SVHI-10 score was 1 (95% CI, -1 to 2) compared with 3 (95% CI, 0-4) after placebo. The median difference between differences at 72 hours between placebo and vitamin B12injections were 1.5 (95% CI, -2 to 5) for the SVHI-10, 1 (95% CI, -9 to 9) for the VFI, and -1 (95% CI, -3 to 2) for the EASE. The improvements after both injections failed to reach the estimated minimal clinically important difference. Of the 20 participants, 4 (20%) reached the estimated minimal clinically important difference in their SVHI-10 score after 72 hours for both vitamin B12and placebo injections. Conclusions and Relevance: This randomized, double-blind, placebo-controlled, crossover trial found that after empirical vitamin B12injection to improve mild voice-related symptoms, the improvement in self-reported voice measures in singers shows no meaningful difference compared with placebo. Trial Registration: ClinicalTrials.gov Identifier: NCT03437824.