Acute mountain sickness (AMS) is a benign and self-limiting syndrome but can progress to life-threatening conditions if leave untreated. This study aimed to assess the efficacy of acetazolamide for the prophylaxis of AMS and disclose potential factors that affect the treatment effect of acetazolamide. Randomized controlled trials comparing the use of acetazolamide versus placebo for the prevention of AMS were included. The incidence of AMS was the primary endpoint. Meta-regression analysis was conducted to explore potential factors associated with acetazolamide efficacy. Trial sequential analysis (TSA) was conducted to estimate the statistical power of the available data. A total of 22 trials were included. Acetazolamide at 125, 250, and 375 mg/ twice daily (bid) significantly reduced incidence of AMS compared to placebo. TAS indicated that the current evidence was adequate confirming the efficacy of acetazolamide at 125, 250, and 375 mg/bid in lowering incidence of AMS. There was no evidence of an association between efficacy and dose of acetazolamide, timing at start of acetazolamide treatment, mode of ascent, AMS assessment score, timing of AMS assessment, baseline altitude, and endpoint altitude. Acetazolamide is effective prophylaxis for the prevention of AMS in doses of 125, 250, and 375 mg/bid. Future investigations should focus on personal characteristics, disclosing the correlation between acetazolamide efficacy and body mass, height, degree of prior acclimatization, individual inborn susceptibility, and history of AMS. Copyright © 2021. Published by Elsevier Inc.