Targeting nucleotide biosynthesis is a proven strategy for the treatment of cancer but is limited by toxicity, reflecting the fundamental nucleotide requirement of dividing cells. The rate limiting step in de novo pyrimidine synthesis is of interest, being catalyzed by two homologous enzymes, CTP synthase 1 (CTPS1) and CTPS2, that could be differentially targeted. Herein, analyses of publicly available datasets identified an essential role for CTPS1 in multiple myeloma (MM), linking high expression of CTPS1 (but not CTPS2) with advanced disease and poor outcomes. In cellular experiments, CTPS1 knockout induced apoptosis of MM cell lines. Exposure of MM cells to STP-B, a novel and highly selective pharmacological inhibitor of CTPS1, inhibited proliferation, induced S phase arrest and led to cell death by apoptosis. Mechanistically, CTPS1 inhibition by STP-B activated DNA damage response (DDR) pathways and induced double-strand DNA breaks which accumulated in early S phase. Combination of STP-B with pharmacological inhibitors of key components of the DDR pathway (ATR, CHEK1 or WEE1) resulted in synergistic growth inhibition and early apoptosis. Taken together, these findings identify CTPS1 as a promising new target in MM, either alone or in combination with DDR pathway inhibition.