Dysregulation of mitochondrial pathways is implicated in several diseases, including cancer. Notably, mitochondrial respiration and mitochondrial biogenesis are favored in some invasive cancer cells, such as osteosarcoma. Hence, the aim of the current work was to investigate the effects of 2-methoxyestradiol (2-ME), a potent anticancer agent, on the mitochondrial biogenesis of osteosarcoma cells. Here, we show that L-lactate induces the mitochondrial biogenesis pathway via up-regulation of COXI. 2-ME inhibited mitochondrial biogenesis via regulation of PGC-1α, COXI, and SIRT3 in a concentration-dependent manner as a consequence of nuclear recruitment of neuronal nitric oxide synthase and nitric oxide generation. It was also proved that 2-ME inhibited SIRT3 activity by binding to both the canonical and allosteric inhibitor binding sites. Moreover, regardless of the mitochondrial biogenesis pathway, 2-ME affected the expression of SDHA. We conclude that mitochondrial biogenesis pathway regulation and SDHA are presented as novel targets of 2-ME, and moreover, that 2-ME may serve as a potent inhibitor of SIRT3. L-lactate was confirmed to exert pro-carcinogenic effects on osteosarcoma cells via the induction of the mitochondrial biogenesis pathway. Thus, L-lactate level may be considered as a prognostic biomarker for osteosarcoma.