Di(2-ethylhexyl) phthalate (DEHP) is recognised as a pollutant with multiple health risks. In this study, multi-omics approaches were used to examine the alterations in immunity, gut microbiota and metabolome, and liver transcriptome in the rats with DEHP-induced subacute liver injury. Following short-term subacute DEHP exposure, the rats exhibited decreased body weight, increased liver weight, impaired liver function and immunity, and signs of liver injury. DEHP exposure reduced the richness, diversity, and evenness of gut microbiota, resulting in elevated levels of Lactobacillus, Romboutsia, and Alistipes and decreased levels of unclassified Muribaculaceae, Oscillibacter, and Akkermansia in the intestine. Multiple gut metabolic pathways were altered by DEHP, among which sphingolipid metabolism was enriched with the most differentially expressed metabolites. In the liver tissues of rats exposed to DEHP, lipid metabolism-related pathways were altered, including downregulated steroid biosynthesis and upregulated fatty acid degradation. In conclusion, the relevant findings suggest that DEHP can cause immune alteration, gut microbiota dysbiosis, gut metabolome disruption, liver transcriptome dysregulation, and result in liver injury in rats. These results could benefit the clinical diagnosis of DEHP-induced subacute liver injury.