Globally, colon cancer is a predominant cause of increased morbidity and mortality annually; therefore, in addition to traditional treatments, new protocols are under continuous investigation. Nanotechnology-based cancer therapy is a new strategy and considered one of the most promising research directions for colon cancer. In this study, we used a silver nanoparticle (AgNP)-based methodology to treat colon cancer cells, and single cell approaches to examine how AgNPs exerted inhibiting effects on cells. We found that AgNPs could apparently destroy cytoskeleton and topography structures, alter cell membrane nanostructures, and thus increase membrane roughness, and depress cell membrane adhesion properties and cell stiffness. We also found that AgNPs caused mitochondrial dysfunctions including hyperpolarization of membrane potential and reactive oxygen species (ROS) accumulation. Notably, AgNPs altered all phenotypes or functions of cells in a dose-dependent manner. Therefore, our research provided a new paradigm for revealing killing mechanisms of AgNPs against colon cancer cells from single cell biophysical aspects, which could advance AgNP-based nanotechnology cancer therapy.