The root system of a plant is essential for plant growth and development because it absorbs nutrients and water from the soil. The root stem cells are maintained and replenished by the quiescent center and are essential for the formation of the specific root structure. In Arabidopsis, the WUSCHEL-RELATED HOMEOBOX 5 (WOX5) plays a key role in regulating root stem cell fate. However, which factor can directly modulate the stability of WOX5 protein remains totally unknown. Here, we report that the peptidase DA1 (LARGE IN CHINESE) interacts with and cleaves WOX5, resulting in the destabilization of WOX5. Genetic analyses support that DA1 acts through WOX5 to regulate root stem cell function. We further demonstrate that cytokinin (CK) signaling induces the accumulation of DA1 protein, thereby decreasing the abundance of WOX5 protein in the root. Consistent with these results, the mutation in DA1 increases the layers of columella stem cells and influences CK-induced differentiation of columella stem cells. Thus, our results reveal a previously unrecognized mechanism by which the cleavage of WOX5 by DA1 connects CK signaling and root stem cell function in Arabidopsis.