This review discusses the mechanisms of non-homologous DNA end joining (NHEJ) and alternative pathways for repairing double-strand breaks (DSBs) in mammalian cells. NHEJ is the primary pathway for DSB repair in all mammalian cells, particularly during the cell cycle. It involves proteins that recognize, process, and ligate DNA ends, allowing for the repair of DSBs even when the ends are not perfectly matched. The flexibility of NHEJ allows it to function on a wide range of DNA-end configurations, though the repaired DNA may contain mutations. The review highlights the roles of key NHEJ proteins, including Ku, DNA-PKcs, Artemis, and XRCC4–DNA ligase IV, in different DNA-end configurations. It also discusses the shunting of DNA-end repair to alternative pathways such as alternative end joining (a-EJ) and single-strand annealing (SSA), and their relevance to human disease. NHEJ is essential for repairing DSBs that occur during DNA replication, ionizing radiation, and other DNA-damaging events. The process involves end resection, where short regions of DNA ends are degraded to generate microhomology, which facilitates end joining. Artemis, a nuclease, plays a critical role in this process, particularly in generating microhomology for end joining. The DNA-PKcs complex is involved in activating Artemis and promoting end resection. The ligase complex, including XRCC4 and DNA ligase IV, is responsible for the final ligation of DNA ends. The review also discusses the roles of other NHEJ proteins, such as DNA polymerases Pol μ and Pol λ, which are involved in template-dependent and -independent DNA synthesis during NHEJ. The XLF and PAXX subpathways are also discussed, as they support ligation by the ligase IV complex. The review highlights the importance of NHEJ in V(D)J recombination and immunoglobulin class switch recombination, and its role in human disease, including severe combined immunodeficiency (SCID). The review also discusses the influence of the cell cycle on NHEJ and alternative pathways. During the G1 phase, NHEJ is favored due to the high abundance of Ku and the suppression of extensive end resection. In S and G2 phases, alternative pathways such as a-EJ and SSA are more active. The review concludes that many questions remain about the mechanisms of DNA repair and the roles of different proteins in NHEJ and alternative pathways. Future studies are needed to fully understand the complexities of DNA repair and its implications for human health.This review discusses the mechanisms of non-homologous DNA end joining (NHEJ) and alternative pathways for repairing double-strand breaks (DSBs) in mammalian cells. NHEJ is the primary pathway for DSB repair in all mammalian cells, particularly during the cell cycle. It involves proteins that recognize, process, and ligate DNA ends, allowing for the repair of DSBs even when the ends are not perfectly matched. The flexibility of NHEJ allows it to function on a wide range of DNA-end configurations, though the repaired DNA may contain mutations. The review highlights the roles of key NHEJ proteins, including Ku, DNA-PKcs, Artemis, and XRCC4–DNA ligase IV, in different DNA-end configurations. It also discusses the shunting of DNA-end repair to alternative pathways such as alternative end joining (a-EJ) and single-strand annealing (SSA), and their relevance to human disease. NHEJ is essential for repairing DSBs that occur during DNA replication, ionizing radiation, and other DNA-damaging events. The process involves end resection, where short regions of DNA ends are degraded to generate microhomology, which facilitates end joining. Artemis, a nuclease, plays a critical role in this process, particularly in generating microhomology for end joining. The DNA-PKcs complex is involved in activating Artemis and promoting end resection. The ligase complex, including XRCC4 and DNA ligase IV, is responsible for the final ligation of DNA ends. The review also discusses the roles of other NHEJ proteins, such as DNA polymerases Pol μ and Pol λ, which are involved in template-dependent and -independent DNA synthesis during NHEJ. The XLF and PAXX subpathways are also discussed, as they support ligation by the ligase IV complex. The review highlights the importance of NHEJ in V(D)J recombination and immunoglobulin class switch recombination, and its role in human disease, including severe combined immunodeficiency (SCID). The review also discusses the influence of the cell cycle on NHEJ and alternative pathways. During the G1 phase, NHEJ is favored due to the high abundance of Ku and the suppression of extensive end resection. In S and G2 phases, alternative pathways such as a-EJ and SSA are more active. The review concludes that many questions remain about the mechanisms of DNA repair and the roles of different proteins in NHEJ and alternative pathways. Future studies are needed to fully understand the complexities of DNA repair and its implications for human health.