The yeast cyclic-AMP dependent protein kinase A (PKA) is a ubiquitous serine-threonine kinase, encompassing three catalytic (Tpk1-3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the catalytic subunit, tpk1, reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ regulator (nej1), co-function in the same pathway. Chromatin immunoprecipitation and resection data suggest that the recruitment of repair proteins and DNA resection is influenced by tpk1 deletion. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair, in addition to nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (the human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.