The role of CREB in LPS signaling is controversial. The objective of this study was to evaluate the effect of LPS on phosphorylation and transcriptional activation of CREB, in comparison to isoproterenol, a β-adrenergic receptor agonist. We show here that LPS elevates intra-cellular cAMP level in RAW264.7 macrophages, with slower kinetics and lower magnitude than isoproterenol. The two agents stimulated CREB phosphorylation on Ser-133 to a similar extent, but with a different mechanism; rapid and mostly PKA-mediated for isoproterenol; slow and MSK1-mediated for LPS. Interestingly, LPS-stimulated phosphorylation of CREB did not result in transcriptional activation of a CRE-regulated luciferase reporter, in contrast to stimulation by isoproterenol. Furthermore, inhibitors of p38 and MSK1, but not PKA, completely blocked the production of IL-10 and TNFα in LPS-stimulated macrophages. Distinctively, the PKA inhibitor H89 blocked the suppressive effect of isoproterenol on TNFα production, as well as its stimulatory effect on IL-10 induction, in LPS-stimulated macrophages. Likewise, while over-expression of dominant negative CREB had no effect on LPS-stimulated TNFα production, it blocked the suppressive effect of isoproterenol on TNFα production in the LPS-stimulated macrophages. Our results thus indicate that PKA-mediated phosphorylation of CREB promotes TNFα suppression and IL-10 induction, whereas the same phosphorylation event initiated by LPS and mediated by MSK1 is non-functional for transcriptional modulation.