Context: Despite the usage of Nerium oleander L. (Apocynaceae) for anticancer studies and traditional remediation, the regulatory effect of N. oleander leaf distillate on cholesterol metabolism is not disclosed sufficiently. Objective: Cholesterol is an important biological molecule and the synthesis rate is regulated by the amount of cholesterol uptake from the diet. The aim of this study was to investigate the regulation of cholesterol metabolism in response to a high-fat diet (HFD) and the effects of N. oleander leaf distillate-supplemented diet (NOHFD) in rats. Materials and methods: Microarray technology was used to clarify the regulation of cholesterol mechanism in HFD and NOHFD-fed rats (375μg/0.5mL distilled water applied by gavage). The treatment period was 90 days. Rat liver tissues were used for microarray analysis using the Affymetrix GeneChip Rat Genome platform. Results of groups were statistically analyzed with the Partek 6.6 bioinformatic program. Results: The HFD group exhibited alterations in the expression levels of about 1945 genes with respect to the normal diet (ND) group. The results showed that expression levels of 47 genes were altered related to cholesterol metabolism in HFD and NOHFD groups. The expression levels of seven genes in the NOHFD group were significantly closer to those in the ND group than those of the HFD group. Discussion and conclusion: To conclude, findings suggest that N. oleander leaf distillate-supplemented food has considerable beneficial effects on cholesterol metabolism-related gene expression levels. © 2014 Informa Healthcare USA, Inc.