Mitochondrial DNA mutations are currently investigated as modifying factors impinging on tumor growth and aggressiveness, having been found in virtually all cancer types and most commonly affecting genes encoding mitochondrial complex I subunits. However, it is still unclear whether they exert a pro- or anti-tumorigenic effect. We here analyzed the impact of three homoplasmic mtDNA mutations (m.3460G>A/MT-ND1, m.3571insC/MT-ND1 and m.3243A>G/MT-TL1) on osteosarcoma progression, chosen since they induce different degrees of oxidative phosphorylation impairment. In fact, the m.3460G>A/MT-ND1 mutation caused only a reduction of complex I activity, whereas the m.3571insC/MT-ND1 and the m.3243A>G/MT-TL1 mutations induced a severe structural and functional complex I alteration. As a consequence, this severe complex I dysfunction determined an energetic defect associated with a compensatory increase in glycolytic metabolism and AMP-activated protein kinase activation. Osteosarcoma cells carrying such marked complex I impairment displayed a reduced tumorigenic potential both in vitro and in vivo, when compared to cells with mild complex I dysfunction, suggesting that mtDNA mutations may display diverse impact on tumorigenic potential depending on the type and severity of the resulting oxidative phosphorylation dysfunction. The modulation of tumor growth was independent from reactive oxygen species production but correlated with hypoxia inducible factor 1α stabilization, indicating that structural and functional integrity of complex I and oxidative phosphorylation are required for hypoxic adaptation and tumor progression.