Dietary phytosterols exert hypocholesterolemic effects by inhibiting cholesterol absorption in the small intestine. However, oxidised phytosterols exert harmful effects. In this study, we compared the effects of dietary stigmasterol or oxidised stigmasterol on cholesterol absorption and metabolism in mice. ICR male mice were fed one of the following diets: a standard AIN diet; the standard diet plus 0.25% cholesterol; the standard diet plus 0.25% cholesterol and 0.25% stigmasterol; or the standard diet plus 0.25% cholesterol and 0.25% oxidised stigmasterol. Stigmasterol, but not oxidised stigmasterol, decreased plasma total cholesterol (TC) levels. Unlike stigmasterol, dietary oxidised stigmasterol increased the cholesterol levels in micellar solutions. Thus, oxidised stigmasterol could not exert hypocholesterolemic effects as it could not displace cholesterol in micellar solutions. In contrast, dietary oxidised stigmasterol downregulates the mRNA expression of genes involved in cholesterol synthesis and upregulates the mRNA expression of genes involved in cholesterol catabolism in mice fed cholesterol. In addition, dietary stigmasterol and oxidised stigmasterol increased the levels of faecal-neutral steroids by downregulating the mRNA expression of Niemann-Pick C1-like 1 protein (NPC1L1) in the small intestine. Dietary stigmasterol may directly regulate the mRNA expression of NPC1L1, whereas dietary oxidised stigmasterol may reduce the mRNA expression of sterol regulatory element-binding protein 2 (SREBP2) and act as a Liver X receptor α (LXRα) agonist, reducing the mRNA expression of NPC1L1. Therefore, oxidised stigmasterol may affect cholesterol absorption and metabolism through a mechanism different from that of stigmasterol.