Loss of TGF-β-induced growth inhibition is a hallmark of many human tumors. Previous studies implied that activation of the anaphase-promoting complex (APC/cyclosome) is involved in the TGF-β signaling pathway, which facilitates the destruction of SnoN, a transcriptional co-suppressor, which leads in turn to the transactivation of TGF-β-responsive genes for cell cycle arrest. The function of APC was demonstrated in TGF-β signal transduction, but the mechanism by which it is activated in response to TGF-β signaling remains unclear. We report here that phosphorylation of Cdc27, a core subunit of APC, in response to TGF-β signaling can facilitate the activation of APC. We have demonstrated that casein kinase II (CKII) is involved in the phosphorylation of Cdc27 in response to TGF-β signaling. Depletion of CKII by shRNA abolishes the TGF-β-induced phosphorylation of Cdc27 and subsequent degradation of SnoN. Disruptive mutation of Cdc27 (S154A) attenuates TGF-β-induced SnoN degradation. In addition, expression of a phosphorylation-resistant Cdc27 mutant significantly attenuates TGF-β-induced growth inhibition. Together, the results suggest that phosphorylation of Cdc27 by CKII is involved in TGF-β-induced activation of APC.