Deinococcus radiodurans RNA ligase (DraRnl) seals 3΄-OH/5΄-PO4 nicks in duplex nucleic acids in which the 3΄-OH nick terminus consists of two or more ribonucleotides. DraRnl exemplifies a widely distributed Rnl5 family of nick-sealing RNA ligases, the physiological functions of which are uncharted. Here we show via gene knockout that whereas DraRnl is inessential for growth of D. radiodurans, its absence sensitizes the bacterium to killing by ionizing radiation (IR). DraRnl protein is present in exponentially growing and stationary phase cells, but is depleted during the early stages of recovery from 10 kGy of IR and subsequently replenished during the late phase of post-IR genome reassembly. Absence of DraRnl elicts a delay in reconstitution of the 10 kGy IR-shattered D. radiodurans replicons that correlates with the timing of DraRnl replenishment in wild-type cells. Complementation with a catalytically dead mutant highlights that nick sealing activity is important for the radioprotective function of DraRnl. Our findings suggest a scenario in which DraRnl acts at genomic nicks resulting from gap-filling by a ribonucleotide-incorporating repair polymerase.