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CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides are a class of small molecules involved in plant growth and development. Although radish (Raphanus sativus) is an important root vegetable crop worldwide, the functions of CLE peptides in its taproot formation remain elusive. Here, a total of 48 RsCLE genes were identified from the radish genome. RNA in situ hybridization showed that RsCLE22a gene was highly expressed in the vascular cambium. Overexpression of RsCLE22a inhibited root growth by impairing stem cell proliferation in Arabidopsis, and radish plants with exogenous supplementation of RsCLE22 peptide (CLE22p) showed a similar phenotype. The vascular cambial activity was increased in RsCLE22a -silenced plants. Transcriptome analysis revealed that CLE22p altered the expression of several genes involved in meristem development and hormone signal transduction in radish. Immunolocalization results showed that CLE22p increased auxin accumulation in vascular cambium. Yeast one-hybrid and dual-luciferase assays showed that the WUSCHEL-RELATED HOMEOBOX 4 (RsWOX4) binds to RsCLE22a promoter and activates its transcription. The expression level of RsWOX4 was related to vascular cambial activity and was regulated by auxin. Furthermore, a RsCLE22a–RsWOX4 module is proposed to regulate taproot vascular cambium activity through an auxin signaling-related pathway in radish. These findings provide novel insights into the regulation of root growth in a horticultural crop. [ABSTRACT FROM AUTHOR]