Summary: REV7, also named MAD2B or MAD2L2, is a subunit of the DNA translesion polymerase zeta and also part of the 53BP1-shieldin complex, which is present at sites of DNA double-strand breaks. REV7 has high sequence similarity to the MAD2 spindle assembly checkpoint protein, prompting us to examine whether REV7 has a checkpoint function. We observed that, in chicken and human cells exposed to agents that induce DNA replication stress, REV7 inhibits mitotic entry; this effect is most evident when the canonical DNA replication stress checkpoint, mediated by ATR, is inhibited. Similar to MAD2, REV7 undergoes conformational changes upon ligand binding, and its checkpoint function depends on its ability to homodimerize and bind its ligands. Notably, even in unchallenged cells, deletion of the REV7 gene leads to premature mitotic entry, raising the possibility that the REV7 checkpoint monitors ongoing DNA replication.