Notch signaling plays a fundamental role in the regulation of diverse biological processes. In the absence of Notch signaling, DNA-bound CSL [CBF1/Su(H)/Lag-1] factor represses gene expression via the direct recruitment of corepressor proteins, including silencing mediator of retinoid and thyroid receptor (SMRT) and KyoT2. Structural studies have unveiled the molecular basis of these interactions in which corepressors bind to CSL using a conserved ΦWΦP motif (Φ: any hydrophobic residues). Here, we comparatively analyzed the CSL-binding motifs of SMRT (¹⁸³⁰EHAPIWRP¹⁸³⁷) and KyoT2 (¹⁸⁶VKAPVWWP¹⁹³) using swap mutants. Various in vivo and in vitro binding assays confirmed that V186 and W192 residues of KyoT2 are major determinants for high affinity binding to CSL. We designed a peptide with the chimeric sequence (VHAPIWWP) of CSL-binding motifs of SMRT and KyoT2. Intriguingly, this peptide acts as an in vivo competitor of endogenous corepressor proteins and an artificial activator for the transcriptional activity of NICD.