Kanglaite, a compound predominantly composed of polyunsaturated fatty acids (PUFAs), has been employed in the clinical treatment of adenocarcinoma non-small cell lung cancer (NSCLC) in China for decades. However, its therapeutic efficacy and specific mechanism in the treatment of squamous NSCLC remains unexplored. In this study, we demonstrate that the co-treatment with ferric ion significantly enhances the cytotoxic effects of kanglaite by inducing ferroptosis in NCL-H1703, a cell line of human lung squamous cell carcinoma. Mechanistic investigations reveal that kanglaite induces mitochondrial dysfunction resulting in reactive oxygen species (ROS) excessive production, which is critical for the induction of ferroptosis. Further analysis shows that kanglaite suppresses the PI3K/AKT signaling pathway, leading to increased IP3 generation. IP3 subsequently binds to and activates IP3R, an endoplasmic reticulum (ER) calcium channel, exacerbating the excessive calcium transfer from the ER to mitochondria. The overloaded mitochondrial calcium contributes to its dysfunction and elevates ROS production. To optimize the synergistic effects of ferric ion and kanglaite, we develop a mesoporous silica-based nanodrug delivery system co-loaded with Kanglaite and Fe3O4, which offers several notable advantages, including reduced drug dosage and a faster therapeutic onset. Finally, in an NCL-H1703 xenograft model, the DMSN/Fe3O4-Kanglaite nanodrug significantly inhibited tumor growth. In conclusion, we identified the function and mechanism of kanglaite in treatment of squamous NSCLC and have developed a DMSN/Fe3O4-Kanglaite nanodrug, providing a superior therapeutic approach for the treatment of squamous NSCLC.Copyright © 2024. Published by Elsevier B.V.