SLC25A51 is a mitochondrial NAD+ transporter that supports oxidative metabolism and cell proliferation. In acute myeloid leukemia (AML), elevated SLC25A51 expression correlates with poor patient outcomes. Depletion of SLC25A51 reduces mitochondrial NAD+ levels, increases apoptosis, and limits AML cell proliferation in vitro and in vivo. Metabolic flux analysis shows that SLC25A51 depletion shifts metabolic flux away from oxidative pathways, reducing TCA cycle activity and promoting reductive flux. SLC25A51 depletion also sensitizes AML cells to glutamine deprivation and enhances the efficacy of 5-azacytidine treatment. These findings suggest that SLC25A51 is a critical regulator of mitochondrial NAD+/NADH ratios, which is essential for AML cell proliferation. Targeting SLC25A51 may represent a therapeutic strategy for refractory AML by disrupting mitochondrial metabolism and promoting cell death. The study highlights the importance of mitochondrial NAD+ in cancer metabolism and provides a rationale for further exploration of SLC25A51 as a therapeutic target in various cancers.SLC25A51 is a mitochondrial NAD+ transporter that supports oxidative metabolism and cell proliferation. In acute myeloid leukemia (AML), elevated SLC25A51 expression correlates with poor patient outcomes. Depletion of SLC25A51 reduces mitochondrial NAD+ levels, increases apoptosis, and limits AML cell proliferation in vitro and in vivo. Metabolic flux analysis shows that SLC25A51 depletion shifts metabolic flux away from oxidative pathways, reducing TCA cycle activity and promoting reductive flux. SLC25A51 depletion also sensitizes AML cells to glutamine deprivation and enhances the efficacy of 5-azacytidine treatment. These findings suggest that SLC25A51 is a critical regulator of mitochondrial NAD+/NADH ratios, which is essential for AML cell proliferation. Targeting SLC25A51 may represent a therapeutic strategy for refractory AML by disrupting mitochondrial metabolism and promoting cell death. The study highlights the importance of mitochondrial NAD+ in cancer metabolism and provides a rationale for further exploration of SLC25A51 as a therapeutic target in various cancers.