Chitosan-stabilized selenium nanoparticles (Ch-SeNPs) are promising agents for cancer therapy due to their unique physicochemical properties, including spherical morphology and uniform size distribution. This study investigates the molecular mechanisms underlying their antitumoral effects, with a focus on the nuclear proteome. Quantitative proteomic analysis revealed 343 nuclear proteins, 47 of which showed significant changes following Ch-SeNPs treatment. Key regulators such as CDK1 and CDC5 were implicated in cell cycle arrest and tumor suppression pathways. Ch-SeNPs also affected processes including mRNA metabolism and cytoskeleton organization. In addition, Ch-SeNPs significantly inhibited tumor growth in a murine melanoma model, supporting their therapeutic potential.