De novo pyrimidine biosynthesis is achieved by cytosolic enzymes, CAD and UMPS, and mitochondrial DHODH. However, how these enzymes are orchestrated remains enigmatical. Here, we show that cytosolic aspartate-producing GOT1 clusters with CAD and UMPS. This cytosolic cluster then connects with DHODH, which is mediated by the mitochondrial outer membrane channel protein VDAC3. Therefore, these proteins form a multi-enzyme complex, named “pyrimidinosome”, involving AMPK as a regulator. Activated AMPK dissociates from the complex, enhancing pyrimidinosome assembly to up-regulate intermediate orotate synthesis, which promotes DHODH-mediated ferroptosis defense. In contrast, pyrimidinosome-mediated UMP biosynthesis is significantly increased in cells lacking AMPK, so that cancer cells with lower expression of AMPK are more reliant on de novo pyrimidine biosynthesis and more vulnerable to its inhibition in vitro and in vivo. Our findings reveal the role of pyrimidinosome in regulating pyrimidine flux and ferroptosis, and suggest a pharmaceutical strategy of targeting pyrimidinosome in cancer treatment.