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Cancer and diabetes, the two mitochondria‐related diseases, have recently been linked to silent mating‐type information regulation 2 homolog 3 (SIRT3) activity irregularities. In this study, the effect of metformin, an antidiabetic with anticancer properties, has been evaluated on mitochondrial functionality markers, cell death pathways, and SIRT3 enzyme activity in the colon cancer cell line, HT‐29, and human embryonic kidney cells (HEK 293). HT‐29 cells were treated with metformin (5, 10, 20, 40, and 80 µM) for 24, 48, and 72 h for measuring the IC₅₀ concentration. Reactive oxygen species (ROS) production, apoptosis, mitochondrial membrane potential, SIRT3 activity, and expression were evaluated against the colon cancer cell line, HT‐29. Results indicated a higher ROS production at 6 than 12 h with metformin treatment. Metformin modified the mitochondrial membrane potential, resulting in cell death …