Activation of the Wnt/beta-catenin signaling pathway by the inhibition of glycogen synthase kinase-3 (GSK3) will induce Tcf7l1 protein degradation to effectively promote embryonic stem cell (ESC) self-renewal. However, the exact mechanism remains unclear. Here, we found that inhibition of casein kinase 2 (Csnk2) by TBB or DMAT was sufficient to block the reduction of the Tcf7l1 protein induced by CHIR99021, a specific inhibitor of GSK-3. Similarly, downregulation of Csnk2 increased the Tcf7l1 level. In contrast, overexpression of Csnk2 significantly decreased Tcf7l1 protein stability in mouse ESCs. Notably, Csnk2 alpha 1 controls Tcf7l1 turnover to a greater degree than the other two isoforms of Csnk2, Csnk2 alpha 2 and Csnk2 beta, as Csnk2 alpha 1-overexpressing mouse ESCs exhibited the lowest level of Tcf7l1. Csnk2 alpha 1 interacted with and phosphorylated Tcf7l1. In addition, the association of Csnk2 alpha 1 and Tcf7l1 was enhanced by CHIR99021. Our study demonstrated, for the first time, that Csnk2 is involved in Tcf7l1 turnover mediated by the Wnt/beta-catenin signaling pathway. These results expand our understanding of the function and circuit of Wnt/beta-catenin signaling pathway in ESCs. (C) 2020 Elsevier Inc. All rights reserved.