This study focused on the modification of lentinan (LNT) and its degradation product, dLNT, through the nitric acid-sodium selenite method, resulting in the synthesis of 4 distinct seleno-polysaccharide Se-LNTs. The selenium moiety was found to be connected to the C6 position of LNT in the form of selenate. The selenization process led to a notable reduction in molecular weight and an augmentation in chain rigidity. As the molecular weight decreased and chain rigidity increased, the cellular uptake of Se-LNTs diminished. Additionally, the uptake pathway transitioned from macropinocytosis to caveolin-mediated endocytosis (CVME). In vitro cell experiments showed that all four Se-LNTs showed obvious anti-tumor activity, and Se-LNT-2 had higher selenium content and cellular uptake rate, showing better inhibitory effect. Furthermore, Se-LNTs were observed to induce a substantial production of reactive oxygen species (ROS) in HCT116 cells. The excessive ROS levels triggered mitochondrial peroxidation damage, escalating mitochondrial inner membrane permeability. This cascade event eventually led to the activation of caspase-3, ultimately leading to apoptosis in HCT116 cells and substantiating the anti-tumor effects of Se-LNTs. The comprehensive investigation into the structural modifications, cellular uptake mechanisms and biological activities of Se-LNTs underscores their potential as promising agents for anti-tumor applications.