The activation of p38α is mediated by its upstream kinase and associated proteins. Here we identify a new nuclear protein, NP60, which regulates the activation of p38α in response to sorbitol treatment. NP60 specifically binds to p38α, but not to JNK and ERK, in vitro and in vivo. Co-transfection of NP60 leads to the phosphorylation and activation of p38α, and subsequently results in the phosphorylation and activation of activating transcription factor 2. The phosphorylation of p38α induced by NP60 requires upstream activity of p38α MAP kinase, MAP kinase kinase 6 (MKK6) or MKK4. Our results indicate that NP60 mediates stress activation of p38α and regulates p38α signaling in a specific way.