AbstractChemoresistance represents a major challenge for osteosarcoma treatment. Despite the improved knowledge of cancer biology, the core determinants of cisplatin (DDP) resistance in osteosarcoma remain unclear and deserve further exploration. Here, RFWD3 is identified as a key regulator of DDP sensitivity in osteosarcoma using a genome‐wide CRISPR screen. It is demonstrated that RFWD3 is overexpressed in post‐chemotherapy osteosarcoma tissues compared to pre‐chemotherapy tissues. Knocking out RFWD3 increased the sensitivity of osteosarcoma cells to DDP treatment. Mechanistically, RFWD3 bound to and ubiquitinated PHGDH at the Lys137 residue, promoting its degradation and conserving cellular oxidized nicotinamide adenine dinucleotide (NAD+). The resulting surplus of NAD+ enhanced the TCA cycle, leading to increased production of aspartic acid and glutamic acid for de novo nucleotide biosynthesis. In addition, virtual screening techniques are employed to identify Lomitapide as a specific inhibitor of the RFWD3‐PHGDH interaction, capable of disrupting the binding between RFWD3 and PHGDH. It is found that Lomitapide exhibits a significant synergistic anti‐osteosarcoma effect when combined with DDP. In conclusion, a specific role of RFWD3 in regulating nucleotide metabolism is revealed and comprised of targetable candidates for overcoming chemoresistance in osteosarcoma.