Both human GH (hGH) and a lipolytic fragment (AOD9604) synthesized from its C-terminus are capable of inducing weight loss and increasing lipolytic sensitivity following long-term treatment in mice. One mechanism by which this may occur is through an interaction with the β-adrenergic pathway, particularly with the β3-adrenergic receptors (β3-AR). Here we describe how hGH and AOD9604 can reduce body weight and body fat in obese mice following 14 d of chronic ip administration. These results correlate with increases in the level of expression of β3-AR RNA, the major lipolytic receptor found in fat cells. Importantly, both hGH and AOD9604 are capable of increasing the repressed levels of β3-AR RNA in obese mice to levels comparable with those in lean mice. The importance of β3-AR was verified when long-term treatment with hGH and AOD9604 in β3-AR knock-out mice failed to produce the change in body weight and increase in lipolysis that was observed in wild-type control mice. However, in an acute experiment, AOD9604 was capable of increasing energy expenditure and fat oxidation in the β3-AR knock-out mice. In conclusion, this study demonstrates that the lipolytic actions of both hGH and AOD9604 are not mediated directly through the β3-AR although both compounds increase β3-AR expression, which may subsequently contribute to enhanced lipolytic sensitivity.