Objective: To establish a Pin1-knockout neural stem cell (NSC) model using the CRISPR/Cas9 system, investigate the impact of Pin1 deficiency on the proliferation of embryonic NSCs, and elucidate the underlying molecular mechanism involving the Notch1 signaling pathway. Methods: A Cas9/sgRNA targeting vector specific to exon 2 of the mouse Pin1 gene was constructed and transfected into C17.2 NSCs. Bi-allelic Pin1 knockout C17.2 colonies were obtained via G418 screening and verified by Sanger sequencing. The knockout efficiency and stemness maintenance (Nestin expression) were validated by immunofluorescence and Western blot analyses. Cell proliferation was assessed using BrdU incorporation and Ki67 staining. RNA-seq was performed to identify differentially expressed genes (DEGs) and enriched signaling pathways, followed by validation of Notch1 pathway activation via qRT-PCR and Western blot. Finally, rescue experiments using the γ-secretase inhibitor DAPT were conducted to confirm the causal link between Notch1 signaling and the observed proliferation phenotype. Results: Three Pin1-null NSC colonies were successfully established, showing complete depletion of the Pin1 protein. These cells maintained Nestin expression but exhibited a significantly hyper-proliferative phenotype, as indicated by increased BrdU incorporation and Ki67 positivity compared to wild-type NSCs (P < 0.05). Transcriptomic analysis revealed aberrant activation of the Notch1 signaling pathway in Pin1-deficient cells, characterized by upregulated Notch1 protein and Hes1 mRNA levels. Crucially, pharmacological inhibition of the Notch pathway with DAPT effectively reversed the upregulation of Notch1 and restored cell proliferation to wild-type levels. Conclusion: Pin1 functions as a negative regulator in C17.2 NSCs by suppressing the Notch1 signaling pathway to prevent excessive cell proliferation. These findings uncover a novel role of Pin1 in embryonic neural development, providing a theoretical basis for further mechanistic exploration.