Folate metabolism plays an essential role in tumor development. Various cancers display therapeutic response to reagents targeting key enzymes of the folate cycle, but obtain chemoresistance later. Therefore, novel targets in folate metabolism are highly demanded. Methylenetetrahydrofolate dehydrogenase/methylenetetrahydrofolate cyclohydrolase 2 (MTHFD2) is one of the key enzymes in folate metabolism and its expression is highly increased in multiple human cancers. However, the underlying mechanism that regulates MTHFD2 expression remains unknown. Here, we elucidate that SIRT4 deacetylates the conserved lysine 50 (K50) residue in MTHFD2. K50 deacetylation destabilizes MTHFD2 by elevating cullin 3 E3 ligase-mediated proteasomal degradation in response to stressful stimuli of folate deprivation, leading to suppression of nicotinamide adenine dinucleotide phosphate production in tumor cells and accumulation of intracellular reactive oxygen species, which in turn inhibits the growth of breast cancer cells. Collectively, our study reveals that SIRT4 senses folate availability to control MTHFD2 K50 acetylation and its protein stability, bridging nutrient/folate stress and cellular redox to act on cancer cell growth.