DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or genetic alterations. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), with defects in these pathways causing genome instability and promoting tumorigenesis. DSBs arise from both endogenous and exogenous sources, including reactive oxygen species, collapsed replication forks, nucleases, ionizing radiation, and chemicals used in cancer therapy. The balance between NHEJ and HR pathways varies among species, cell types, and cell cycle phases. Regulatory factors that influence DSB repair pathway choice include the expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins function exclusively in one pathway, some proteins, such as MRE11/RAD50/NBS1 (XRS2), BRCA1, histone H2AX, PARP-1, RAD18, DNA-PKcs, and ATM, influence both pathways. DNA-PKcs plays a role in mammalian NHEJ and also influences HR through a complex regulatory network involving crosstalk with ATM and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM. The regulation of DSB repair pathway choice is crucial for maintaining genome stability and preventing cancer.DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or genetic alterations. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), with defects in these pathways causing genome instability and promoting tumorigenesis. DSBs arise from both endogenous and exogenous sources, including reactive oxygen species, collapsed replication forks, nucleases, ionizing radiation, and chemicals used in cancer therapy. The balance between NHEJ and HR pathways varies among species, cell types, and cell cycle phases. Regulatory factors that influence DSB repair pathway choice include the expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins function exclusively in one pathway, some proteins, such as MRE11/RAD50/NBS1 (XRS2), BRCA1, histone H2AX, PARP-1, RAD18, DNA-PKcs, and ATM, influence both pathways. DNA-PKcs plays a role in mammalian NHEJ and also influences HR through a complex regulatory network involving crosstalk with ATM and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM. The regulation of DSB repair pathway choice is crucial for maintaining genome stability and preventing cancer.