p38α plays an important role in many inflammatory diseases, such as skin inflammation, endotoxic shock and arthritis. Ubiquitination is a vital posttranslational modification of proteins and plays a crucial regulatory role in inflammatory cells. It has been reported that ubiquitination of Tak1 and TAB1 upstream of p38α can regulate p38α activation respectively. However, p38α ubiquitination is not yet clear. In this paper, we showed that E3 ubiquitin ligase Nedd4 is a regulatory component of the p38α pathway and is responsible for polyubiquitination of p38α through K48-linked and K63-linked polyubiquitination. The levels of p38α and its downstream target TNF-α were increased in Nedd4 deficient macrophages response to LPS compared with wild-type cells. AP-1 activity and degradation of p38α were induced by Nedd4 in a dose-dependent manner. Furthermore, we found that phosphorylation of p38α is involved in the interactions between p38α and Nedd4 and subsequently promotes polyubiquitination of p38α, especially K48-linked polyubiquitination by Nedd4. The different conformation of two p38α isoforms (p38αV1 and p38αV2) might be the cause of their different interactions with Nedd4 and their polyubiquitination sites by Nedd4. Thus, NEDD4 is a previously unknown component of the p38α signaling complex necessary for TNF-α activation.