Metformin, a widely used anti-diabetic drug, has been shown to inhibit cancer progression by targeting mitochondrial complex I in cancer cells. This study demonstrates that metformin reduces tumor growth by inhibiting mitochondrial complex I activity, which is essential for cellular respiration and energy production in cancer cells. Metformin inhibits cellular respiration and proliferation in the presence of glucose but induces cell death when glucose is absent, indicating that cancer cells rely on glycolysis for survival in the presence of metformin. Metformin also reduces hypoxic activation of hypoxia-inducible factor 1 (HIF-1), which is crucial for tumor survival under low-oxygen conditions. These effects were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed, suggesting that metformin's anti-cancer effects are dependent on its ability to inhibit mitochondrial complex I. In vivo, metformin treatment inhibited the growth of control human cancer cells but not those expressing NDI1, confirming that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on mitochondrial complex I inhibition. Metformin's ability to inhibit complex I was also demonstrated in other cancer cell lines, including A549 cells. The study further shows that metformin reversibly inhibits mitochondrial complex I, which is essential for maintaining mitochondrial membrane potential and ATP production. Metformin's effects on mitochondrial complex I were distinct from those of rotenone, an irreversible complex I inhibitor. Metformin's inhibition of complex I reduces tumor growth by inducing cell death under glucose deprivation and inhibiting HIF-1 activation, which is critical for tumor survival under hypoxic conditions. These findings highlight the importance of mitochondrial complex I inhibition in cancer progression and suggest that metformin could be an effective treatment for cancers that rely on mitochondrial function for survival. The study also indicates that metformin's anti-cancer effects are not limited to mitochondrial function but may also involve its ability to reduce insulin levels, which are known to promote cancer growth. Overall, the study provides strong evidence that metformin inhibits cancer progression by targeting mitochondrial complex I in cancer cells.Metformin, a widely used anti-diabetic drug, has been shown to inhibit cancer progression by targeting mitochondrial complex I in cancer cells. This study demonstrates that metformin reduces tumor growth by inhibiting mitochondrial complex I activity, which is essential for cellular respiration and energy production in cancer cells. Metformin inhibits cellular respiration and proliferation in the presence of glucose but induces cell death when glucose is absent, indicating that cancer cells rely on glycolysis for survival in the presence of metformin. Metformin also reduces hypoxic activation of hypoxia-inducible factor 1 (HIF-1), which is crucial for tumor survival under low-oxygen conditions. These effects were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed, suggesting that metformin's anti-cancer effects are dependent on its ability to inhibit mitochondrial complex I. In vivo, metformin treatment inhibited the growth of control human cancer cells but not those expressing NDI1, confirming that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on mitochondrial complex I inhibition. Metformin's ability to inhibit complex I was also demonstrated in other cancer cell lines, including A549 cells. The study further shows that metformin reversibly inhibits mitochondrial complex I, which is essential for maintaining mitochondrial membrane potential and ATP production. Metformin's effects on mitochondrial complex I were distinct from those of rotenone, an irreversible complex I inhibitor. Metformin's inhibition of complex I reduces tumor growth by inducing cell death under glucose deprivation and inhibiting HIF-1 activation, which is critical for tumor survival under hypoxic conditions. These findings highlight the importance of mitochondrial complex I inhibition in cancer progression and suggest that metformin could be an effective treatment for cancers that rely on mitochondrial function for survival. The study also indicates that metformin's anti-cancer effects are not limited to mitochondrial function but may also involve its ability to reduce insulin levels, which are known to promote cancer growth. Overall, the study provides strong evidence that metformin inhibits cancer progression by targeting mitochondrial complex I in cancer cells.