The purpose of this study was to determine the effects of phytosterol ester-enriched vegetable oil on the serum concentration of cholesterol and to assess its safety in healthy subjects with normocholesterolemics. Twenty-two healthy subjects completed this study in a double blind, parallel arm design. The subjects were randomly divided into 2 groups: control group (n = 11) and phytosterol ester-enriched group (n = 11), and fed either 42 g of phytosterol ester-enriched vegetable oil or control vegetable oil, containing about 1.336 g and 0.117 g of phytosterol (as the major free sterol) respectively, daily for 4 weeks. Serum lipids, apolipoproteins, serum fat-soluble vitamins, serum phytosterols and biochemical parameters were measured. The subjects were asked to report any side effect. Phytosterol ester-enriched vegetable oil significantly reduced the concentrations of serum total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B, by 3.3%, 4.4% and 3.2% respectively, compared with control oil. No significant changes were found in the concentrations of triacylglycerol, high-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, remnant-like lipoprotein cholesterol and other kinds of apolipoproteins. Neither vegetable oil affected the concentrations of fat-soluble vitamins (retinol and α-tocopherol) or β-carotene. Serum β-sitosterol and campesterol were significantly higher in the phytosterol ester-enriched vegetable oil group compared with the control oil group. The concentrations of biochemical parameters did not change in either group. There were no side effects or serious adverse events in either group during the administration. In conclusion, the results showed that a daily 1.336 g intake of phytosterol (as the major free sterol) is effective in lowering serum total- and LDL-cholesterol in healthy men and has no negative physiologic action.