Substantial evidence has linked dehydroepiandrosterone (DHEA) levels to the anti-obesity and antidiabetic effects of exercise. While 5 '-adenosine monophosphate-activated protein kinase (AMPK) is a negative regulator of adipocyte differentiation and lipid accumulation, activation of mammalian target of rapamycin complex 1 (mTORC1), which is inhibited by AMPK, is required for adipocyte differentiation and positively regulates lipid accumulation. DHEA treatment activates the AMPK pathway in C2C12 myotubes. Hence, DHEA addition to preadipocytes and adipocytes might activate AMPK and inhibit mTORC1, resulting in the inhibition of adipogenesis and lipid accumulation. Therefore, we investigated the effect of DHEA on the AMPK pathway, mTORC1 activity, adipocyte differentiation, and lipid accumulation in 3T3-L1 cells. DHEA suppressed lipid accumulation and adipogenic marker expression during differentiation. It also activated AMPK signaling in preadipocytes and adipocytes and suppressed mTORC1 activity during differentiation. These results suggest that the activation of the AMPK pathway and inhibition of mTORC1 activity may mediate the anti-obesity effect of DHEA, providing novel molecular-level insights into its physiological functions. (C) 2020 Elsevier Inc. All rights reserved.