AU-rich elements (AREs) in the 3'-untranslated region (UTR) of unstable mRNA dictate their degradation or mediate translational repression. Cell signaling through p38alpha MAPK is necessary for post-transcriptional regulation of many pro-inflammatory cytokines. Here, the cis-acting elements of interleukin-6 (IL-6) 3'-UTR mRNA that required p38alpha signaling for mRNA stability and translation were identified. Using mouse embryonic fibroblasts (MEFs) derived from p38alpha(+/+) and p38alpha(-/-) mice, we observed that p38alpha is obligatory for the IL-1-induced IL-6 biosynthesis. IL-6 mRNA stability is promoted by p38alpha via 3'-UTR. To understand the mechanism of cis-elements regulated by p38alpha at post-transcriptional level, truncation of 3'-UTR and the full-length 3'-UTR with individual AUUUA motif mutation placed in gene reporter system was employed. Mutation-based screen performed in p38alpha(+/+) and p38alpha(-/-) mouse embryonic fibroblast cells revealed that ARE1, ARE2, and ARE5 in IL-6 3'-UTR were targeted by p38alpha, and truncation-based screen showed that IL-6 3'-UTR-(56-173) was targeted by p38alpha to stable mRNA. RNA secondary structure analysis indicated that modulated reporter gene expression was consistent with predicted secondary structure changes.