The endogenous thyroid hormone (TH) metabolite 3,5-diiodo-l-thyronine (3,5-T₂) acts as a metabolically active substance affecting whole-body energy metabolism and hepatic lipid handling in a desirable manner. Considering possible adverse effects regarding thyromimetic action of 3,5-T₂treatment in rodents, the current literature remains largely controversial. To obtain further insights into molecular mechanisms and to identify novel target genes of 3,5-T₂in liver, we performed a microarray-based liver tissue transcriptome analysis of male lean and diet-induced obese euthyroid mice treated for 4 weeks with a dose of 2.5 µg/g bw 3,5-T₂. Our results revealed that 3,5-T₂modulates the expression of genes encoding Phase I and Phase II enzymes as well as Phase III transporters, which play central roles in metabolism and detoxification of xenobiotics. Additionally, 3,5-T₂changes the expression of TH responsive genes, suggesting a thyromimetic action of 3,5-T₂in mouse liver. Interestingly, 3,5-T₂in obese but not in lean mice influences the expression of genes relevant for cholesterol and steroid biosynthesis, suggesting a novel role of 3,5-T₂in steroid metabolism of obese mice. We concluded that treatment with 3,5-T₂in lean and diet-induced obese male mice alters the expression of genes encoding hepatic xenobiotic-metabolizing enzymes that play a substantial role in catabolism and inactivation of xenobiotics and TH and are also involved in hepatic steroid and lipid metabolism. The administration of this high dose of 3,5-T₂might exert adverse hepatic effects. Accordingly, the conceivable use of 3,5-T₂as pharmacological hypolipidemic agent should be considered with caution.