The effects of high-dose folic acid (FA) supplementation in healthy individuals on blood folate concentrations and immune response are unknown. The aim of the study was to evaluate the effects of daily consumption of a tablet containing 5 mg FA on serum folate; number and cytotoxicity of natural killer (NK) cells; mRNA expression of dihydrofolate reductase (), methylenetetrahydrofolate reductase (), interferon γ (), tumor necrosis factor α (), and interleukin 8 () genes; and concentrations of serum inflammatory markers. This prospective clinical trial was conducted in 30 healthy Brazilian adults (15 women), aged 27.7 y (95% CI: 26.4, 29.1 y), with a body mass index (in kg/m) of 23.1 (95% CI: 22.0, 24.3). Blood was collected at baseline and after 45 and 90 d of the intervention. Serum folate concentrations were measured by microbiological assay and HPLC-tandem mass spectrometry [folate forms, including unmetabolized folic acid (UMFA)]. We used real-time polymerase chain reaction to assess mononuclear leukocyte mRNA expression and flow cytometry to measure the number and cytotoxicity of NK cells. Serum folate concentrations increased by ∼5-fold after the intervention ( < 0.001), and UMFA concentrations increased by 11.9- and 5.9-fold at 45 and 90 d, respectively, when compared with baseline ( < 0.001). UMFA concentrations increased (>1.12 nmol/L) in 29 (96.6%) participants at day 45 and in 26 (86.7%) participants at day 90. We observed significant reductions in the number ( < 0.001) and cytotoxicity ( = 0.003) of NK cells after 45 and 90 d. Compared with baseline, mRNA expression was higher at 90 d ( = 0.006) and and mRNA expressions were higher at 45 and 90 d ( = 0.001 for both). This noncontrolled intervention showed that healthy adults responded to a high-dose FA supplement with increased UMFA concentrations, changes in cytokine mRNA expression, and reduced number and cytotoxicity of NK cells. This trial was registered at www.ensaiosclinicos.gov.br as RBR-2pr7zp.