In addition to the known role of PA in mTOR signaling, it has been suggested that PA regulates epidermal growth factor receptor (EGFR) trafficking from the membranes towards the nucleus and vice versa, and regulates EGFR expression in the nucleus, thus controlling EGF signaling 26. Although generally PA is reported to maintain mTOR complexes in a steady-state condition 24, PLD1-produced PA has been shown to specifically bind to the FRB domain of mTOR 19 and displace an endogenous mTOR inhibitor, DEP domain-containing mTOR-interacting protein (DEPTOR), to activate mTORC1 25. PLD-produced PA is unique in that it contains fatty acid chains with one or two degrees of unsaturation 23. Of the several enzymes that are involved in PA biogenesis, PLD1 is the enzyme responsible for activating mTOR via either mitogen or amino acid stimulation 19, 20, 21, 22. PA is a critical regulator of mTOR signaling 19. Phospholipase D (PLD) hydrolyzes phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline 18. Despite this, mTOR also maintains homeostasis of adipogenesis by suppressing the expression of PPARγ through insulin receptor substrate-1 (IRS-1)/Akt signaling 16, 17, suggesting an indispensable function for mTOR in adipogenesis. Conversely, activation of mTORC1 enhances adipogenesis by increasing PPARγ 13, confirming a positive role for mTOR in adipogenesis 15. Inhibition of mTORC1 by either rapamycin treatment or an adipose-specific knockout of regulatory-associated protein of mTOR (also known as RPTOR or raptor, a major component of mTORC1), inhibits adipogenesis 11, 14. Recently, a number of studies have shown that mTOR is involved in adipogenesis and lipid metabolism 10, 11, 12, 13. mTORC1 controls translation and protein synthesis by phosphorylating ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1), whereas mTORC2 activates Akt, serum/glucocorticoid-regulated kinase (SGK), and protein kinase Cα (PKCα) 8, 9. mTOR forms two distinct complexes: mTOR complex1 (mTORC1) and mTOR complex2 (mTORC2) 7. The mammalian target of rapamycin (mTOR) pathway regulates many cellular and developmental processes by responding to growth factors and nutrients 6. Subsequently, these regulators activate PPARγ and C/EBPα, which upregulate each other and maintain their expression to govern the entire adipogenic process by activating additional transcription factors 4. Expression of C/EBPβ and C/EBPδ is induced in preadipocytes during very early differentiation. Adipocyte differentiation is a well-controlled process regulated by an elaborate network of transcription factors, including the CCAAT/enhancer-binding proteins C/EBPβ, C/EBPδ, C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ) 4, 5. An understanding of the molecular mechanisms that regulate adipogenesis is required to reduce obesity and the accompanying susceptibility to many diseases. Dietary changes towards high protein and high fat intake have raised the prevalence of obesity over the last decade, increasing the risk of many disorders such as diabetes mellitus, hyperlipidemia, insulin resistance, cardiovascular disease, and cancer 1, 2, 3. Obesity is defined by excessive accumulation of white adipose tissue above the normal level of adipocyte differentiation owing to an energy imbalance. Taken together, our findings provide convincing evidence for a direct role of PLD1 in adipogenic differentiation by regulating IRS-1 phosphorylation at serine 636/639 through DEPTOR displacement and mTOR activation. Further investigation revealed that PA displaces DEP domain-containing mTOR-interacting protein (DEPTOR) from mTORC1, which subsequently phosphorylates insulin receptor substrate-1 (IRS-1) at serine 636/639 in 3T3-L1 cells. Moreover, the elevated differentiation in PLD1-knockdown 3T3-L1 cells was reduced by either PA treatment or PLD1 expression, confirming negative roles of PLD1 and PA in adipogenic differentiation. Conversely, treatment with phosphatidic acid (PA), a PLD product, and overexpression of PLD1 both caused a decrease in adipogenic differentiation. We also found that differentiation of 3T3-L1 preadipocytes was enhanced by the depletion of PLD1 levels or inhibition of PLD1 activity by VU0155069, a PLD1-specific inhibitor. In line with this observation, PLD activity decreased in both high fat diet (HFD)-fed mice and ob/ob mice. We showed that PLD activity was downregulated by both 3-Isobutyl-1-methylxanthine (IBMX) and insulin upon induction of differentiation in 3T3-L1 adipogenic cells. In the present study, we identified PLD1 as a negative regulator of adipogenic differentiation. Phospholipase D1 (PLD1) plays a known role in several differentiation processes, but its role in adipogenic differentiation remains unknown.