The impact of sitosterol formulation particle size on the intestinal sterol absorption and the sterol status in various tissues in Dunkin Hartley guinea pigs was investigated. Three groups of animals (six each) were fed a basal diet (“control”) or a basal diet containing either customary sitosterol (“customary”, particle size: 10 000–90 000 nm) or nanoscale sitosterol (“nanoscale”, particle size: 200–300 nm). The average daily sitosterol intake was 21 ± 7 mg (control), 154 ± 8 mg (customary), and 127 ± 18 mg (nanoscale) for 2 weeks. Sitosterol and cholesterol were analyzed in samples of plasma, blood cells, bile, liver, kidney, jejunal mucosa/serosa, cecum, colon and feces. Concentrations of sitosterol in all analyzed matrices increased significantly in the supplemented groups when compared to control group. No differences in the sitosterol concentrations in analyzed matrices occurred between nanoscale and customary group. The cholesterol concentrations in tissues remained unchanged. Fecal fatty acid and sterol distributions were modified during sitosterol intervention. Both particle sizes equally increased sitosterol levels in cholesterol-metabolizing compartments in the guinea pig. No differences in body compartment accumulation and intestinal absorption of the different sitosterol particle sizes were observed.