The existence of cancer stem cells is widely acknowledged as the underlying cause for the challenging curability and high relapse rates observed in various tumor types, including non-small cell lung cancer (NSCLC). Despite extensive research on numerous therapeutic targets for NSCLC treatment, the strategies to effectively combat NSCLC stemness and achieve a definitive cure are still not well defined. The primary objective of this study was to examine the underlying mechanism through which Fructose-1,6-bisphosphatase 1 (FBP1), a gluconeogenic enzyme, functions as a tumor suppressor to regulate the stemness of NSCLC. Herein, we showed that overexpression of FBP1 led to a decrease in the proportion of CD133-positive cells, weakened tumorigenicity, and decreased expression of stemness factors. FBP1 inhibited the activation of Notch signaling, while it had no impact on the transcription level of Notch 1 intracellular domain (NICD1). Instead, FBP1 interacted with NICD1 and the E3 ubiquitin ligase FBXW7 to facilitate the degradation of NICD1 through the ubiquitin–proteasome pathway, which is independent of the metabolic enzymatic activity of FBP1. The aforementioned studies suggest that targeting the FBP1-FBXW7-NICD1 axis holds promise as a therapeutic approach for addressing the challenges of NSCLC recurrence and drug resistance.