This study was performed to evaluate whether increasing hemoglobin before ascent by prophylactic erythropoietin injections prevents acute mountain sickness (AMS). This open-label, randomized, controlled trial involved 39 healthy volunteers with hemoglobin ≤15.5 g/dL who were divided randomly into erythropoietin (n=20) and control (n=19) groups. Epoetin alpha 10,000 IU injections were given weekly for four consecutive weeks. On day 1, and 7 days after the last injection (day 29), oxygen saturation (SaO2), and hemoglobin were measured. The subjects departed Seoul on day 30 and arrived at Annapurna base camp (ABC, 4,130 m) on day 34. AMS was diagnosed when headache and Lake Louise score (LLS) of ≥3 were present. Immediate descent criteria followed US Army recommendations. Two groups differ in hemoglobin levels on day 29 (15.4±1.1 vs 14.2±1.0 g/dL, P=0.001). At ABC, erythropoietin group had a significantly lower mean LLS, AMS incidence, and number of subjects who met immediate descent criteria. Multiple logistic regression analysis showed that SaO2<87% and control group, but not hemoglobin<15.0 g/dL, independently predicted satisfaction of immediate descent criteria. Erythropoietin-related adverse effects were not observed. In conclusion, erythropoietin may be an effective prophylaxis for AMS.(Clinical Trial Registry Number; NCT 01665781).Graphical Abstract