Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break repair pathways in mammalian cells and is required for both V (D) J recombination and class switch recombination (CSR), two Ig gene–diversification processes occurring during B cell development. DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a component of the classical NHEJ machinery and has a critical function during V (D) J recombination. However, its role in CSR has been controversial. In this study, we examined the pattern of recombination junctions from in vivo–switched B cells from two DNA-PKcs–deficient patients. One of them harbored mutations that did not affect DNA-PKcs kinase activity but caused impaired Artemis activation; the second patient had mutations resulting in diminished DNA-PKcs protein expression and kinase activity. These results were compared with those from DNA-PKcs–deficient mouse B cells. A shift toward the microhomology-based alternative end-joining at the recombination junctions was observed in both human and mouse B cells, suggesting that the classical NHEJ pathway is impaired during CSR when DNA-PKcs is defective. Furthermore, cells from the second patient showed additional or more severe alterations in CSR and/or NHEJ, which may suggest that DNA-PKcs and/or its kinase activity have additional, Artemis-independent functions during these processes.