Recent reports demonstrated that 3,5-diiodo-L-thyronine (T2) was able to prevent lipid accumulation in the liver of rats fed a high fat diet (HFD). In the present study, we investigated how the rat liver responds to HFD and T2 treatment by assessing the transcription profiles of some genes involved in three processes of lipid metabolism: oxidation, storage and secretion. By using real-time RT-PCR we assessed the mRNA levels of the peroxisome proliferator-activated receptors (PPAR-a-g-d), and of their target enzymes acyl-CoA oxidase (AOX), and stearoyl CoA desaturase (SCD-1). Moreover, we analyzed the expression of the adipose triglyceride lipase (ATGL) involved in lipid mobilization, of the main PAT proteins acting in lipid droplet (LD) turnover, and of apoprotein B (apoB), the major protein component of very low density lipoproteins (VLDL). Overall, our data demonstrated that T2 administration to HFD rats counteracts most of the hepatic transcriptional changes that occurred in response to the excess exogenous fat. In particular, our results suggest that T2 may prevent the pathways leading to lipid storage in LDs, promote the processes of lipid mobilization from LDs and secretion as VLDL, in addition to the stimulation of pathways of lipid oxidation. In conclusion, our findings might get an insight into the mechanisms underlying the anti-steatotic ability of T2 and support the therapeutic potential role of T2 for preventing or treating liver steatosis.