Di(2-ethylhexyl) phthalate (DEHP) is widely used and has been implicated in hepatotoxicity, although the mechanism is unclear. Here, we investigated the effect of DEHP on hepatic cholesterol metabolism in SD rats exposed to 0 and 300 mg/kg/day DEHP for 12 weeks. An RNA-Seq analysis was performed to describe the hepatic responses to long-term DEHP exposure in combination with serological and oxidative stress parameter measurements. DEHP increased the serum levels of total cholesterol (TC), high-density lipoprotein (HDL), and alanine transaminase (ALT). Moreover, DEHP increased the content of malondialdehyde (MDA) and decreased antioxidant enzyme activities in the liver. Transcriptomic results revealed that DEHP dramatically changed the cholesterol metabolism pathway and oxidation-reduction process and depressed gene expression involved in cholesterol efflux and monooxygenase activity. Total antioxidant capacity (T-AOC) positively correlated with Abcg5 and Abcg8. Overall, this study showed the mechanisms underlying hepatotoxicity caused by DEHP, providing new insights into understanding DEHP poisoning. • Effects of DEHP on the transcriptome in liver were studied. • DEHP increased cholesterol and HDL-C in serum. • Transcriptomics data revealed DEHP affected genes related to cholesterol efflux. • DEHP induced oxidative damage in rat liver. • DEHP caused disturbed cholesterol homeostasis contributed to oxidative stress. [ABSTRACT FROM AUTHOR]