We generated preadipocyte cell lines impaired in adrenomedullin production through integration of an adrenomedullin small interfering RNA expression vector. The reduction of adrenomedullin synthesis strongly accelerated adipose differentiation. These results were bolstered when overexpression of active adrenomedullin peptide led to delayed differentiation. Therefore, we propose that adrenomedullin is an antiadipogenic factor. Moreover, we checked whether insulin, a proadipogenic factor, regulates expression of adrenomedullin. We observed that insulin had an inhibitory effect on adrenomedullin expression in isolated human adipocyte cells. This response was dose dependent and was reversed by resistin, a new anti-insulin agent. We quantified circulating adrenomedullin in healthy obese patients and observed a threefold increase of adrenomedullin compared with lean patients. Furthermore, adrenomedullin plasma levels are negatively correlated to plasma insulin levels in these obese patients. The insulin inhibitory response was also observed in vivo in Sprague-Dawley rats but not in the insulin-resistant Zucker rat, suggesting that adrenomedullin expression is upregulated in insulin-resistant adipose cells. Using adrenomedullin promoter-luciferase reporter gene constructs, we have shown that the adrenomedullin response to insulin is mediated by insulin-responsive elements. These findings provide new insight into fat mass development and the relationship between obesity and elevated circulating adrenomedullin levels in diabetic patients.