Objective: To evaluate both efficacy and safety in humans of long-term consumption of spreads containing plant sterol esters. Design: Randomized double-blind placebo-controlled parallel trial. Subjects: Hundred and eighty-five healthy volunteers (35-64y). Intervention: Volunteers daily consumed 20g spread enriched with 1.6g plant sterols as fatty acid esters or a control spread for 1 y. They continued their habitual diet and lifestyle. Outcome measures included efficacy markers such as total and LDL-cholesterol, a large range of safety parameters, and reporting of adverse events. Results: Consumption of the plant sterol ester-enriched spread consistently lowered total and LDL cholesterol during the 1 y period on average by 4 and 6%, respectively (0.01 < P < 0.05). Plant sterols intake did on average not result in a lower carotenoid concentration (when expressed per LDL-cholesterol) after 52 weeks (P > 0.05). However, carotenoid concentrations changed over time. Plant sterols intake reduced lipid adjusted α- and β-carotene-concentrations by only 15-25% after 1 y, relative to control. Lipid-adjusted fat-soluble vitamin concentrations remained unchanged. Plant sterol concentrations in serum were increased from 2.76 to 5.31 (μmol/mmol total cholesterol) for campesterol (P < 0.0001) and from 1.86 to 2.47 (μmol/mmol total cholesterol) for β-sitosterol (P < 0.0001). The increase in total plant sterol concentration in red blood cells (5.29-9.62 μg/g) did not affect red blood cell deformability. Hormone levels in males (free and total testosterone) and females (luteinizing hormone, follicle stimulating hormone, β-estradiol and progesterone) as well as all clinical chemical and hematological parameters measured were unaffected. Adverse events reported were not different between subjects consuming control spread and subjects consuming plant sterol esters-enriched spread. Conclusion: Consumption of a plant sterol esters-enriched spread is an effective way to consistently lower blood cholesterol concentrations and is safe to use over a long period of time.