To reduce the nonspecific cytotoxicity of 5-fluorouracil (5-FU) and enhance its selectivity towards cancer cells, we conjugated 5-FU with F16 ((E)-4-(1H-indol-3-ylvinyl)-N-methylpyridinium iodide), a delocalized lipophilic cationic (DLC) compound with mitochondrion-targeting ability, via vulnerable bonds (ester, amido, disulfide) to synthesize four conjugated compounds: F16–5-FU, F16–OOC-FU, F16–NHOC-FU, and F16–SS-FU. Biological evaluations demonstrated that F16–OOC-FU decreased 5-FU's antiproliferative activity on nontumor GES-1 cells, exhibited mitochondrion-targeting in tumor SGC-7901 cells, and induced cell death, cycle arrest, and elevated reactive oxygen species (ROS). F16–SS-FU showed significantly increased cytotoxicity when co-administered with dithiothreitol (DTT). All conjugates inherited F16's fluorescent properties, which were utilized to study cellular uptake and localization. This study provides a novel strategy to enhance 5-FU's selectivity for cancer cells and expands F16's application as a mitochondrion-targeting probe.