Background: The objective of this study was to evaluate the effects of micellar phytosterols on cholesterol absorption in humans. Methods: 24 healthy adults (mean age 37 years, 16 women and 8 men) underwent four cholesterol-absorption tests to compare the effects of micellar phytosterols supplement containing 500, 300, 200 mg phytosterols and placebo on cholesterol absorption in a cross-over design. Individual absorption tests were separated by 2 weeks. Using the single oral isotope method, inhibition of cholesterol absorption was determined by gas-chromatography mass spectrometry measurement of the differences in plasma hexadeuterated cholesterol enrichment 4 days after test meals with or without phytosterols. Each test meal provided 500 kcal and 89 mg cholesterol including 40 mg tracer cholesterol. We used a general linear mixed model with SAS to test the significance of treatment effect. Results: Micellar phytosterols had a significant overall effect on cholesterol absorption (p = 0.0002), reduced cholesterol absorption by 23.3% at a dose of 300 mg phytosterols (p = 0.0004) and by 32.0% at a dose of 500 mg phytosterols (p = 0.0001) compared with the placebo. The effect of 200 mg treatment did not reach statistical significance but there was a tendency (p = 0.052). Conclusion: Water-dispersible, micellar phytosterols reduced cholesterol absorption effectively at very low doses.