<p>Oral squamous cell carcinoma (OSCC) presents ongoing clinical difficulties due to its frequent recurrence and unfavorable prognosis. Betanin (BTN) is recognized for its potent antioxidant and anticancer properties; however, its specific molecular mechanisms, particularly those related to the apoptotic and TGF-β/PI3K-Akt signalling pathways, remain partially unexplored. An in vivo study was performed using 7,12-dimethylbenz[a]anthracene (DMBA)-induced OSCC in hamsters, where the expression patterns of PI3K, Akt, Bax, Bcl-2, Caspase-3, Caspase-9, mutant p53, TGF-β RI &amp; RII, SMAD-2, SMAD-4, and SMAD-7 were examined through immunohistochemistry, Western blot, and qRT-PCR, with a focus on PI3K/Akt and TGF-β signaling pathways. BTN, administered in vivo, attenuated tumor progression in OSCC-afflicted hamsters in a dose-dependent manner, inhibiting growth-regulatory signalling nodes such as PI3K, Akt, Bcl-2, mutant p53, TGF-β RI, TGF-β RII, SMAD-2, and SMAD-4, while promoting the expression of apoptotic proteins SMAD-7, Bax, Caspase-3, and Caspase-9. BTN exhibits multi-targeted anticancer potential in OSCC by modulating the expression of with key apoptotic (Bax, Bcl-2, Caspases-3 &amp; 9, mutant p53) and growth-regulatory signalling nodes PI3K, Akt, TGF-β RI, TGF-β RII, SMAD-2, 4, and 7. Our previous molecular docking study directly engages the aforementioned proteins. As a result, the current experimental framework identifies BTN as a potential chemopreventive agent, meriting additional mechanistic and therapeutic research in the context of oral cancer.</p> Graphical abstract <p>Betanin modulate the DMBA induced TGF-β and PI3K/AKT pathway to induce the apoptosis</p> <p></p>