Y-family DNA polymerases are specialized to copy damaged DNA and are associated with increased mutagenesis due to their low fidelity. It is believed that the mechanism of nucleotide selection by Y-family DNA polymerases involves conformational changes preceding nucleotidyl transfer but there is limited experimental evidence for such structural changes. In particular, nucleotide-induced conformational changes in bacterial or eukaryotic Y-family DNA polymerases have to date not been extensively characterized. Using hydrogen/deuterium exchange mass spectrometry, we demonstrate here that the Escherichia coli Y-family DNA polymerase DinB and its human ortholog DNA polymerase κ undergo a conserved nucleotide-induced conformational change in the presence of undamaged DNA and the correct incoming nucleotide. Notably, this holds true for damaged DNA containing N2-furfuryl-deoxyguanosine that is efficiently copied by these two polymerases, but not for damaged DNA containing the major groove modification O6-methyl-deoxyguanosine that is a poor substrate. Our observations suggest that DinB and pol κ utilize a common mechanism for nucleotide selection involving a conserved pre-chemical conformational transition promoted by the correct nucleotide and only preferred DNA substrates.