BACKGROUND: It is well known that blood donors are at increased risk of iron deficiency and subsequent development of iron deficiency anemia. We aimed to investigate the effect of factors influencing hemoglobin (Hb) levels. STUDY DESIGN AND METHODS: Initiated in 2010, the Danish Blood Donor Study is a population-based study and biobank. We performed multivariable linear regression analysis to assess the effects of donation activity, physiologic and lifestyle factors, and diet on Hb levels among 15,197 donors. We also performed multivariable logistic regression to evaluate the effects of these factors on the risk of having low Hb (defined as Hb below the 10th percentile among men and women, respectively) and of a decrease in Hb greater than 0.5 mmol/L (0.8 g/dL) between successive donations. All analyses were performed stratified for sex and smoking status. We also tested a previously used model for the prediction of Hb. RESULTS: The strongest predictors of Hb and risk of low Hb were low ferritin (<15 ng/mL) and current use of iron supplementation (yes/no). No dietary factors were found to be consistently significant in multivariable models predicting Hb levels, risk of having low Hb, or risk of a decrease in Hb greater than 0.5 mmol/L. We found similar effects to previous studies of factors in the predictive model, with little additional effect of including smoking status and ferritin. CONCLUSIONS: As ferritin was the strongest predictor of Hb, this study supports the implementation of regular ferritin measurement as a method of risk assessment among blood donors.