Triptolide (TP) has been found to have anti‐tumor effects. However, more potential molecular mechanisms of TP in the progression of non‐small cell lung cancer (NSCLC) deserve further investigation. Cell proliferation, apoptosis, invasion, and stemness were detected by cell counting kit 8 assay, EdU assay, flow cytometry, transwell assay, and sphere formation assay. Cell glycolysis was evaluated by corresponding assay kits. 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 2 (PFKFB2) expression was measured by western blot (WB), qRT‐PCR and immunohistochemical staining. PI3K/AKT pathway‐related markers were determined by WB. Besides, xenograft tumor model was conducted to evaluate the anti‐tumor effect of TP in NSCLC. Our results revealed that TP treatment suppressed NSCLC cell proliferation, invasion, stemness, glycolysis, and enhanced apoptosis. PFKFB2 was upregulated in NSCLC tissues and cells, and its expression was decreased by TP. PFKFB2 knockdown restrained NSCLC cell functions, and its overexpression also eliminated TP‐mediated NSCLC cell functions inhibition. TP decreased PFKFB2 expression to inactivate PI3K/AKT pathway. Moreover, PI3K/AKT pathway inhibitor LY294002 also could reverse the promoting effect of PFKFB2 on NSCLC cell functions. In addition, TP suppressed NSCLC tumorigenesis by inhibiting PFKFB2/PI3K/AKT pathway. In conclusion, TP exerted anti‐tumor role in NSCLC, which was achieved by reducing PFKFB2 expression to inactivate PI3K/AKT pathway.