Background <p>Tumor cell differentiation is a critical determinant of malignancy and clinical treatment selection. Pancreatic ductal adenocarcinoma (PDAC), a poorly differentiated and highly aggressive tumor, has a poor prognosis, whereas well-differentiated tumors often correlate with better outcomes. The mechanisms underlying differentiation and its therapeutic potential remain unclear.</p> Objectives <p>This study aims to investigate whether inducing transdifferentiation in pancreatic cancer cells can reduce malignancy, focusing on the role of the transcription factor NeuroD1 and its regulatory pathways.</p> Methods <p>We analyzed single-cell RNA-seq data from the GEO database to identify differentiation-associated genes. NeuroD1 was overexpressed in PDAC cells to assess its effects on transdifferentiation and proliferation. Drug screening and molecular docking were performed to identify differentiation-inducing compounds. RNA sequencing, coimmunoprecipitation, and mass spectrometry were used to identify NeuroD1-interacting proteins. Cell/patient-derived xenograft mouse models are utilized for in vivo experiments and compound efficacy testing.</p> Results <p>Highly differentiated tumor cells exhibited elevated NeuroD1 expression. NeuroD1 overexpression promoted neuronal transdifferentiation and suppressed proliferation. Neuropathiazol, a neurogenic inducer, was found to bind MET and upregulate NeuroD1 via the PI3K/Akt pathway, enhancing transdifferentiation and inhibiting tumor growth. Neurog3 was identified as a functional partner of NeuroD1.</p> Conclusion <p>Our findings demonstrate that pancreatic cancer cells can be induced to transdifferentiate through NeuroD1 activation or pharmacological induction, suggesting a potential therapeutic strategy to mitigate malignancy by reprogramming tumor cells into less aggressive states.</p>