Abstract Background Regular intake of plant sterol (phytosterol)-enriched foods enhances the cholesterol lowering effect of diets. One side effect associated with plant sterol consumption is a modest reduction in plasma carotenoid concentrations. This study investigated the effect of consuming a low-fat National Cholesterol Education Programme (NCEP) Step 1 diet, including a low-fat plant sterol ester (PSE)-enriched spread on cholesterol metabolism to determine if specific dietary advice to increase daily fruit and vegetable intake could prevent reduced plasma carotenoid concentrations. Materials and methods In this randomised, crossover double-blind trial, 48 hypercholesterolaemic men received 21 g day−1 of a low-fat PSE-enriched spread or placebo for 3 weeks, interrupted by 3 weeks washout. Individuals also adhered to a NCEP Step 1 diet and repeated 3-day food diaries monitored adherence. Specific advice was provided to increase dietary fruit and vegetable intakes. Fasting blood samples were collected at pre- and post-intervention for lipoprotein and carotenoid analysis. Results Plasma total and low-density lipoprotein (LDL) cholesterol concentrations were significantly (P < 0.05) reduced, by 4.6 and 7.1%, respectively, after the PSE-enriched low-fat spread. Plasma apo B concentrations were significantly (P < 0.0005) lower after the PSE spread. PSE consumption was also associated with significantly (P < 0.05) lower total plasma β-carotene concentrations, but this change was not significant after lipid standardisation. PSE consumption had no effect on retinol, α-carotene, γ-tocopherol, α-tocopherol, lutein, zeaxanthin, β-crypyoxanthin or lycopene concentrations. Conclusion Dietary advice to increase daily fruit and vegetable consumption may be effective in preventing a reduction in plasma carotenoid concentrations previously associated with PSE consumption. Further, PSE incorporated in a low-fat spread and consumed as part of a NCEP Step 1 diet are effective in reducing total and LDL cholesterol.