Insulin resistance and type 2 diabetes (T2D) are characterized by hyperlipidemia. The aim of the present study was to elucidate whether T2D contributes to abnormal cholesterol (CHOL) homeostasis. Experiments were carried out in the small intestine and liver of Psammomys obesus, a model of nutritionally induced T2D. Our results show that diabetic animals exhibited a lower intestinal CHOL uptake, which was associated with a decrease in 1) the gene and protein expression of Niemann-Pick C1 like 1 that plays a pivotal role in CHOL incorporation in the enterocytes; and 2) mRNA of ATP-binding cassette transporters (ABC)A1 that mediates CHOL efflux from intestinal cells to apolipoprotein A-I and high-density lipoprotein. No changes were observed in the other intestinal transporters scavenger receptor-class B type I (SR-BI) and annexin 2. On the other hand, in diabetic animals, a significant mRNA decrease was noticed in intestinal ABCG5 and ABCG8 responsible for the secretion of absorbed CHOL back into the lumen. Furthermore, jejunal PCSK9 protein was diminished and low-density lipoprotein receptor was raised, along with a significant down-regulation in jejunal 3-hydroxy-3-methylglutaryl-coenzyme A reductase in P. obesus with T2D. Finally, among the transcription factors tested, only an increase in liver X receptors alpha and a decrease in peroxisome proliferator-activated receptors delta/beta mRNAs were detected in the intestine. In the liver, there was 1) an augmentation in the protein mass of Niemann-Pick C1 like 1, SR-BI, and annexin 2; 2) an up-regulation of SR-BI mRNA; 3) a fall in ABCG8 protein content as well as in ABCG5 and ABCA1 mRNA; and 4) an augmentation in liver X receptors alpha and peroxisome proliferator-activated receptors beta/delta mRNA, together with a drop in sterol regulatory element binding protein-2 protein. Our findings show that the development in P. obesus with T2D modifies the whole intraenterocyte and hepatocyte machinery responsible for CHOL homeostasis.