The mechanism underlying anti-inflammatory effects of macrolide antibiotics remains uncertain. In this study, we first show the evidences concerning the possible link between leukocytic cyclic adenosine monophosphate (cAMP) signaling and the mechanism of anti-inflammatory, cytoprotective actions of macrolides. The clinical range of macrolides (i.e., erythromycin, roxithromycin, and clarithromycin) preferentially inhibited nuclear factor-κB activation mediated by reactive oxygen intermediates, inducing cAMP-dependent signaling [i.e., cAMP and cAMP-responsive element-binding protein (CREB)] by “primed” but not “resting” leukocytes. In this context, cAMP/CREB inhibition with adenosine 3′:5′-cyclic monophosphothioate, rp-isomer (rp-cAMPs) and CREB decoy oligonucleotides reduced the anti-inflammatory actions of macrolides. These results thus indicate that macrolide-induced cAMP/CREB signaling, selectively by primed leukocytes, plays a major role in the mechanism of anti-inflammatory actions of macrolides.