Stanyl esters dissolved in margarine inhibit cholesterol absorption, lower sterol absorption in general, and lower serum total cholesterol, LDL-cholesterol and plant sterol levels. To find out whether stanyl esters inhibit absorption of fat-soluble vitamins and β-carotene in acute experiments, we performed two fat-tolerance tests fortified with vitamins (retinol 0.9–3.7 mg, α-tocopherol 70–581 mg), β-carotene (25–150 mg) and squalene (0.5 g) with and without 1 g of stanyl ester added to the test meal in ten healthy men. The concentrations or areas under the curves (AUC) of cholesterol, triacylglycerols, squalene and α-tocopherol, β-carotene and retinyl palmitate showed typical postprandial changes in serum, chylomicrons, VLDL and VLDL infranatant (intermediate-density lipoproteins, LDL and HDL) over 24 h after the test meal without stanyl esters, and they were not affected by the addition of stanyl esters. The post-absorptive serum campesterol concentration and campesterol : cholesterol were significantly lowered at 6–9 h by stanyl ester supplementation, reflecting reduced sterol absorption efficiency. Changes in vitamin and β-carotene AUC did not correlate with the given doses. In conclusion, the present study shows that stanyl esters dissolved in margarine do not detectably interfere in a short-term study with the absorption of α-tocopherol, β-carotene or retinol measured by a 24 h oral fat-load test.