Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a two-stage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, ​NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that ​NPM1 (​nucleophosmin) regulates TLS via interaction with the catalytic core of DNA ​polymerase-η (​polη), and that ​NPM1 deficiency causes a TLS defect due to proteasomal degradation of ​polη. Moreover, the prevalent ​NPM1c+ mutation that causes ​NPM1 mislocalization in ~30% of AML patients results in excessive degradation of ​polη. These results establish the role of ​NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in ​NPM1.