The involvement of the TRP vanilloid 1 (TRPV1) cation channel on the 5‐Fluorouracil (5‐FU)‐caused Ca2+ signals through the activation of the apoptotic signaling pathway and stimulating the mitochondrial Ca2+ and Zn2+ accumulation‐induced reactive oxygen species (ROS) productions in several cancer cells, except the colorectal cancer (HT‐29) cell line, was recently reported. I aimed to investigate the action of silver nanoparticles (SiNPs) and 5‐FU incubations through the activation of TRPV1 on ROS, apoptosis, and cell death in the HT‐29 cell line. The cells were divided into four groups: control, SiNP (100 µM for 48 h), 5‐FU (25 μM for 24 h), and 5‐FU + SiNP. SiNP treatment through TRPV1 activation (via capsaicin) stimulated the oxidant and apoptotic actions of 5‐FU in the cells, whereas they were diminished in the cells by the TRPV1 antagonist (capsazepine) treatment. The apoptotic and cell death actions of 5‐FU were determined by increasing the propidium iodide/Hoechst rate, caspase‐3, ‐8, and ‐9 activations, mitochondrial membrane depolarization, lipid peroxidation, and ROS, but decreasing the glutathione and glutathione peroxidase. The increase of cytosolic free Ca2+ and Zn2+ into mitochondria via the stimulation of TRPV1 current density increased oxidant and apoptotic properties of 5‐FU in the cells. For the therapy of HT‐29 tumor cells, I found that the combination of SiNPs and 5‐FU was synergistic via TRPV1 activation.