Blood donation may influence subsequent NHL development via temporary immune system alterations. To test the hypothesis that frequent blood donation is associated with an increased risk of NHL and its most common histologic subtypes, we followed 36,576 men in the Health Professionals Follow-up Study (HPFS), who provided information on frequency of blood donation in the past 30 years in 1992. We confirmed 544 incident cases of NHL through 2010. Cox proportional hazards regression was used to calculate hazards ratios (HR) and 95% CI for the risk of all NHL and major NHL histologic subtypes associated with number of blood donations. In this prospective study, there was no significant evidence of an association between blood donation frequency and incidence of NHL (age-adjusted HR 1.26, 95% CI 0.94–1.68, comparing >20 donations vs. 0 donations over 30 years, p for trend = 0.18) or of any major NHL subtypes.