Despite its wide distribution in the central nervous system, the presence of Neuropeptide Y (NPY) in peripheral tissues has been detected. White adipose tissue (WAT) is a new site of NPY synthesis and secretion. The development of brown-like adipocytes in WAT is controlled by hypothalamic NPY neurons through interaction with sympathetic nervous system (SNS). However, whether peripheral NPY has a direct effect on induction of the Uncoupling protein1 (UCP1)-positive adipocytes is unknown. We have used adipocytes derived from C3H10T1/2 stem cells as a model of brown-like adipocyte, and investigated the role of NPY in their differentiation and activation. In general, NPY had no effect on brown adipogenesis of C3H10T1/2 stem cell, but suppressed db-cAMP activation of brown-like adipocytes, which was due to blunting brown fat-relevant gene expression and mitochondrial function. NPY showed suppression in a receptor-dependent manner, inhibition of endogenous cAMP production and cAMP-PKA-dependent pathways p38 MAPK and CREB phosphorylation were involved in the downstream mechanisms. A novel role of NPY in the peripheral is presented, which may help decrease energy expenditure in WAT of obese subjects.