CCAAT/enhancer-binding protein (C/EBP) 13 plays an important role in proliferation and differentiation of 3T3-L1 preadipocytes. C/EBP beta is sequentially phosphorylated during the 3T3-L1 adipocyte differentiation program, first by MAPK/Cyclin A/cdk2 on Thr(188) and subsequently by GSK3 beta on Ser(184) or Thr(179). Dual phosphorylation is critical for the gain of DNA binding activity of C/EBP beta. In this manuscript, we found that phosphorylation also contributed to the stability of C/EBP beta. Both ex vivo and in vitro experiments showed that phosphorylation by MAPK/Cyclin A/cdk2 and GSK3 beta protected C/EBP beta from mu-calpain-mediated proteolysis, while phosphorylation on Thr(188) by MAPK/Cyclin A/cdk2 contributed more to the stabilization of C/EBP beta, Further studies indicated that phosphorylation mimic C/EBP beta was insensitive to both calpain accelerator and calpain inhibitor. Thus, phosphorylation might contribute to the stability as well as the gain of DNA binding activity of C/EBP beta. (C) 2012 Elsevier Inc. All rights reserved.