Mitogen-activated protein kinases (MAPKs) play major roles in regulating important physiological processes. MAPK phosphatases (MKPs) dephosphorylate and inhibit MAPKs activity, thereby negatively regulating MAPKs signaling. Altered expression of MKP-1, the most studied member of the MKP family, has been implicated in the development of various kinds of cancers and in the acquisition of chemoresistance. MKP-1 mRNA is labile and has specific AU-rich elements (AREs) in its 3' untranslated region (3'UTR) that direct posttranscriptional gene regulation. In this study, activities of reporters comprised of luciferase coding region and various lengths of MKP-1 3'UTR were analyzed. The results indicated that transcripts bearing complete MKP-1 3'UTR or ARE fragments had a significant lower expression of the reporter gene. And co-expression of tristetraprolin (TTP) further inhibited the expression of reporters. An RNA pull-down assay was established for the identification of MKP-1 RNA-interacting proteins. And RNA-immunoprecipitation assay (RNA-IP) was performed and reporter mRNAs containing MKP-1 AREs were found to be associated with RNA-binding proteins such as TTP and Hu antigen R (HuR). In this paper, we proved that 3'UTR of MKP-1 mRNA contributed largely to the regulation of MKP-1 expression and we provided two practical biochemical assays for identifying ARE-binding proteins associated with MKP-1 mRNA.