Background/Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a high rate of recurrence and a dismal prognosis. Studies have shown that pancreatic cancer stem cells (PCSCs) are a subpopulation that contributes to tumor progression, resistance to therapeutics, and metastasis, making them a key subpopulation to target for treatment. Gedunin (GD), a natural compound derived from Azadirachta indica (neem), has shown anticancer properties in pancreatic cancer cells, but its effects on PCSCs remains unclear. This study evaluated the effects of GD in pancreatic cancer stem cells, highlighting its impacts on tumor growth and progression and focusing on its impact on the sonic hedgehog (Shh) signaling pathway. Methods: Functional assays were performed to assess the effect of GD on the sphere-forming ability, colony formation, and self-renewal of PCSCs. Athymic mice xenograft models were utilized to evaluate the tumor suppression effect of GD in vivo. Furthermore, the anticancer effect of GD on PCSCs was assessed using both in vitro and in vivo limiting dilution assay. GD-induced changes in Shh signaling and key stem cell marker expressions in PCSCs were evaluated. Results: GD effectively inhibited tumor growth in xenograft models and reduced the percentage of PCSCs. GD was effective in decreasing PCSCs’ proliferative, self-renewal, and colony-forming capacity. GD decreased the protein expression levels of key Shh signaling markers Gli1 and Shh, stem cell markers SOX2, Nanog, and Oct4, metastasis-related proteins MMP-2, MMP-3, and MMP-9, and EMT markers Tgf1, Slug, Snail, and Twist in both PDAC cells and PCSCs. We demonstrated a significant decrease in the spheroid formation and self-renewal capacity of the (ALDH+) PCSC population following GD treatment in HPAC cells, indicating its potential antagonistic effects on PCSCs. GD was highly effective in reducing tumor volume, stemness, and metastasis in both early and late chemotherapy. In vivo limiting dilution assay using CD133+/LGR5+ PCSC xenografts demonstrated that GD reduces tumor growth, metastasis, and stemness associated with PCSCs by downregulating the expression of Shh and Gli1. GD treatment also reduced micrometastatic lesions in the lung, liver, and brain, as identified using H&E staining. Conclusions: The findings highlight GD’s potential as a promising therapeutic candidate for PDAC, with the ability to target both bulk tumor cells and PCSCs. By simultaneously suppressing tumor growth, stemness, and metastatic spread, GD may contribute to more effective treatment strategies and improved patient outcomes.