The Peroxisome‐Proliferator‐Activated Receptor‐Gamma Co‐Activator 1 Beta (PGC1β) is a co‐activator of several transcription factors involved in lipid metabolism and very low density lipoprotein (VLDL) secretion. Therefore, PGC1β may be a master regulator of lipid metabolism in liver. To study this hypothesis, human liver HepG2 cells were infected with adenoviruses containing PGC1β or β‐galactosidase (control), and cells and medium were extracted to determine changes in cellular lipids. A wide array of lipids was measured using liquid chromatography/mass spectrometry (LC/MS/MS) using an Applied Biosystems 4000Q Trap. PGC1β over‐expression caused significant decreases in liver cellular neutral lipids. There was a 61.5±3.9% decrease in triacylglycerides (TAG), 40.9±6.7% decrease in diacylglycerides (DAG), and 52.9±3.8% decrease in monoacylglycerides (MAG). During this period, over‐expression of cellular PGC1β decreased the secretion of neutral lipids: 39.7±4.9% decrease in TAG, 60.2±4.1% decrease in DAG; MAG were not detectable in HepG2 medium. There were no significant changes in any of the cellular phospholipid concentrations or species between control cells and cells treated with PGC1β. Our studies show that over‐expression of PGC1β greatly influences neutral lipid metabolism in human liver HepG2 cells.Grant Funding SourceNIH, Rutgers University