Temperature distributions inside a living cell reflect the thermodynamics and functions of cellular components. We used a newly-developed method of mitochondrial thermometry based on Rhodamine B methyl ester, which equilibrates as a thermosensitive mixture of nonfluorescent and fluorescent resonance forms. Prostaglandin E2 (PGE2) is released from hepatic non-parenchymal Kupffer cells and acts as an inflammatory factor to impact various functions of hepatocytes. The activity of PGE2 on energy mechanism of hepatocytes has not been fully elucidated and in particular, which PGE2 receptor mediates the functions has been elusive. We identified EP4 as the major receptor of PGE2 via our mitochondrion-thermometry approach and then substantiated this receptor's role in hepatic metabolism. We discovered that PGE2 is able to decrease intracellular temperature of hepatocytes, via increasing some lipogenic genes' expressions, hampering lipolysis and mitochondrial β-oxidation, reducing intracellular ATP level and elevating cAMP level through EP4 receptor. The redox status of hepatocytes represented by FAD vs FAD + NADH ratio is influenced by PGE2 in an EP4 receptor-dependent manner. Collectively, these data demonstrate that PGE2 regulates metabolism of hepatocytes mainly through EP4 receptor.