Growth hormone (GH) has long been known to stimulate lipolysis and insulin resistance; however, the molecular mechanisms underlying these effects are unknown. In the present study, we demonstrate that GH acutely induces lipolysis in cultured adipocytes. This effect is secondary to the reduced expression of a negative regulator of lipolysis, fat-specific protein 27 (FSP27; aka Cidec) at both the mRNA and protein levels. These effects are mimicked in vivo as transgenic overexpression of GH leads to a reduction of FSP27 expression. Mechanistically, we show GH modulation of FSP27 expression is mediated through activation of both MEK/ERK- and STAT5-dependent intracellular signaling. These two molecular pathways interact to differentially manipulate peroxisome proliferator-activated receptor gamma activity (PPARγ) on the FSP27 promoter. Furthermore, overexpression of FSP27 is sufficient to fully suppress GH-induced lipolysis and insulin resistance in cultured adipocytes. Taken together, these data decipher a molecular mechanism by which GH acutely regulates lipolysis and insulin resistance in adipocytes.