Osteosarcoma is the most common primary malignant bone tumor in adolescents and children and prone to develop lung metastasis. Its prognosis has been virtually unimproved over the last few decades, especially in patients with metastases, who suffer from a dismal survival. Recently, increasing attention has been devoted to monocarboxylate transporters-related (MCTs) metabolic reprogramming. However, the role of MCT1 and MCT4 in osteosarcoma progression and the underlying mechanisms remain to be further elucidated.In this study, we established MCT1 and/or MCT4 knockout cell lines by CRISPR/Cas9 genome editing technology. Then, we assessed glycolysis and oxidative phosphorylation capacities by measuring lactate flux and oxygen consumption. We also performed flowcytometry to test circulating tumor cells and PET/CT to evaluate glucose uptake.MCT1 was found to be involved in both glycolysis and oxidative respiration due to its ability to transport lactate in both directions. MCT1 inhibition significantly reduced circulating tumor cells and distant metastases partially by increasing oxidative stress. MCT4 was primarily related to glycolysis and responsible for lactate export when the concentration of extracellular lactate was high. MCT4 inhibition dramatically suppressed cell proliferation in vitro and impaired tumor growth with reduction of glucose uptake in vivo.Our results demonstrate the functional heterogeneity and redundancy of MCT1 and MCT4 in glucose metabolism and tumor progression in osteosarcoma. Thus, combined inhibition of MCT1 and MCT4 may be a promising therapeutic strategy for treating tumors expressing both transporters.© 2023. The Author(s).