This study explores the pharmacological investigation and anti-cancer potential of folate-modified Gellan gum nanoparticles, focusing on their impact on cancer cells through p53 activation and the inhibition of the mTOR/PI3K pathway. The nanoparticles were meticulously characterized using advanced techniques, including Nuclear Magnetic Resonance (NMR) and Fourier-Transform Infrared Spectroscopy (FTIR) to ensure structural integrity. Additionally, drug release kinetics, particle size, zeta potential, and entrapment efficiency were assessed to understand the formulation's physicochemical properties. Anti-cancer efficacy was evaluated through Sulforhodamine B (SRB) assay, demonstrating a significant reduction in cell viability upon exposure to the folate-modified Gellan gum nanoparticles. The p53 assay revealed an upregulation of the tumor suppressor protein, indicating activation of the intrinsic apoptotic pathway. Concurrently, the mTOR assay demonstrated inhibition of the mTOR/PI3K signaling pathway, further supporting the anti-cancer potential of the nanoparticles. Comprehensive characterization studies corroborated the efficacy findings, with NMR and FTIR confirming the successful modification of Gellan gum. The nanoparticle formulation exhibited controlled drug release, optimal particle size, desirable zeta potential, and high entrapment efficiency, collectively contributing to its potential as an effective anti-cancer agent. In conclusion, this research provides a thorough investigation into the anti-cancer potential of folate-modified Gellan gum nanoparticles. The synergy of SRB, p53, and mTOR assays, coupled with detailed characterization parameters, establishes a comprehensive understanding of the formulation's efficacy. These findings support the development of Gellan gum nanoparticles as a promising candidate for targeted cancer therapy, warranting further exploration and clinical translation.