The lipid lowering effect of niacin has been attributed to the inhibition of cAMP production in adipocytes, thereby inhibiting intracellular lipolysis and release of non-esterified fatty acids (NEFA) to the circulation. However, long term niacin treatment leads to a normalization of plasma NEFA levels and induces insulin resistance, for which the underlying mechanisms are poorly understood. The current study addressed the effects of long term niacin treatment on insulin-mediated inhibition of adipocyte lipolysis and focused on the regulation of cAMP levels. APOE*3-Leiden.CETP transgenic mice treated with niacin for 15 weeks were subjected to an insulin tolerance test and showed whole body insulin resistance. Similarly, adipocytes isolated from niacin treated mice were insulin resistant and, interestingly, exhibited an increased response to cAMP stimulation by 8Br-cAMP, β₁ and β₂-adrenergic stimulation. Gene expression analysis of the insulin and β-adrenergic pathways in adipose tissue indicated that all genes were down-regulated, including the gene encoding the cAMP degrading enzyme phosphodiesterase 3B (PDE3B). In line with this, we showed that insulin induced a lower PDE3B response in adipocytes isolated from niacin treated mice. Inhibiting PDE3B with cilostazol increased lipolytic responsiveness to cAMP stimulation in adipocytes. These data show that long term niacin treatment leads to a down-regulation of PDE3B in adipocytes which could explain part of the observed insulin resistance and the increased responsiveness to cAMP stimulation.