The de novo purine synthesis pathway is fundamental for nucleic acid production and cellular energetics, yet the role of mitochondrial metabolism in modulating this process remains underexplored. In many cancers, metabolic reprogramming supports rapid proliferation and survival, but the specific contributions of the tricarboxylic acid (TCA) cycle enzymes to nucleotide biosynthesis are not fully understood. Here, we demonstrate that the TCA cycle enzyme succinate dehydrogenase (SDH) is essential for maintaining optimal de novo purine synthesis in normal and cancer cells. Genetic or pharmacological inhibition of SDH markedly attenuates purine synthesis, leading to a significant reduction in cell proliferation. Mechanistically, SDH inhibition causes an accumulation of succinate, which directly impairs the purine biosynthetic pathway. In response, cancer cells compensate by upregulating the purine salvage pathway, a metabolic adaptation that represents a potential therapeutic vulnerability. Notably, co-inhibition of SDH and the purine salvage pathway induces pronounced antiproliferative and antitumoral effects in preclinical models. These findings not only reveal a signaling role for mitochondrial succinate in regulating nucleotide metabolism but also provide a promising therapeutic strategy for targeting metabolic dependencies in cancer.