We investigated the effect of thalidomide on transcriptional and post-transcriptional cyclooxygenase-2 (COX-2) expression, including a pathway leading to COX-2 mRNA destabilization. We found that thalidomide inhibited the interleukin-1beta (IL-1beta)-mediated induction of COX-2 protein and mRNA in Caco-2 cells. Transient transfection with a COX-2 promoter construct demonstrated that thalidomide did not affect IL-1beta-induced transcriptional activation of COX-2, although it did decrease the stability of COX-2 mRNA and suppress IL-1beta-induced cytoplasmic shuttling of an mRNA stabilizing protein, HuR. Thalidomide also suppressed IL-1beta-induced p38 mitogen-activated protein kinase (MAPK) activation, while a p38 MAPK inhibitor destabilized COX-2 mRNA and the cytoplasmic shuttling of HuR induced by IL-1beta. These data suggest that one of the molecular mechanisms of thalidomide may be destabilization of COX-2 mRNA through inhibition of cytoplasmic shuttling of HuR and p38 MAPK.