The cyclooxygenase-2 (COX-2) gene encodes the inducible prostaglandin synthase enzyme implicated in inflammation, cell growth, and tumorigenesis. Regulation of the COX-2 gene expression at the post-transcriptional level is poorly understood. For example, protein factors that regulate the post-transcriptional mRNA metabolism of COX-2 have not been fully characterized. In this study, we demonstrate that the RNA-binding protein HuR binds to COX-2 mRNA and regulates its expression. We show that there are three binding sites for HuR in the 3'-untranslated region of human COX-2. These sites are located at the following positions in the COX-2 3'-untranslated region 39-84 nucleotides (nt), 1155-1187 nt, and 1244-1256 nt (hereinafter referred to as Sites I, II and III, respectively). Although all three sites are present in the 4.6-kb COX-2 mRNA, only site I is present in the shorter 2.8-kb isoform. HuR in MDA-MB-231 cell extracts associated with COX-2 mRNA at the identified sites. Further, HuR location in the cytoplasm was induced by serum withdrawal, a stimulus known to induce COX-2 mRNA. Down-regulation of HuR by two independent methods, namely RNA interference as well as antisense RNA expression, significantly attenuated serum withdrawal-induced increase in COX-2 mRNA (both the 4.6- and 2.8-kb isoforms) and protein levels. These data suggest that HuR binding to COX-2 is critical for its post-transcriptional mRNA stabilization.