Adipocyte differentiation is an ordered multistep process requiring the sequential activation of several groups of adipogenic transcription factors, including CCAAT/enhancer-binding protein-alpha and peroxisome proliferator-activated receptor-gamma, and coactivators. Here we show that replication factor C 140, which was known to act as a coactivator for CCAAT/enhancer-binding protein-alpha in our previous study, was phosphorylated on the proliferating cell nuclear antigen-bindng domain during the adipocyte differentiation process. Calmodulin-dependent protein kinase II was responsible for phosphorylating replication factor C 140 in the process of adipocyte differentiation. Ser518 of replication factor C 140 was identified as a major target of calmodulin-dependent protein kinase II phosphorylation in vitro. Calmodulin-dependent protein kinase II inhibitor attenuated phosphorylation of replication factor C 140 by differentiation inducers and blocked replication factor C 140-derived transcriptional activation. Taken together, these findings demonstrate that calmodulin-dependent protein kinase II signaling leads the cooperative transactivation of CCAAT/enhancer-binding protein-alpha and replication factor C 140 through an increase in replication factor C 140 phosphorylation, and subsequently enhances the transcriptional activation of target genes involved in adipocyte differentiation.