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), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous and exogenous sources, and the balance between NHEJ and HR varies among species, cell types, and cell cycle phases. This review discusses the regulatory factors that control DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated 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 NHEJ or HR, several proteins influence both pathways, including the MRE11/RAD50/NBS1 complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ but 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. HR is considered more accurate for DSB repair, using homologous sequences to prime repair synthesis. However, even with perfect homology, repair can be error-prone. HR is more accurate than NHEJ, but both pathways can lead to genome rearrangements. NHEJ is error-prone but can repair DSBs with complementary overhangs. The choice between NHEJ and HR is regulated by factors such as template availability, cell cycle phase, and the functions of specific cell types. In yeast, HR is more efficient in diploids than haploids, while in higher eukaryotes, HR is more efficient in cells with homologous templates. DNA-PKcs and ATM regulate DSB repair pathway choice, with DNA-PKcs playing a key role in NHEJ and ATM in HR. The regulation of DSB repair pathways is crucial for maintaining genome stability and preventing cancer. Understanding these pathways can lead to clinical benefits in cancer therapy, including more effective chemo- and radiotherapy strategies and improved detection of cancer in its earliest stages. Targeting the proteins that regulate the choice between NHEJ and HR can enhance gene targeting and reduce the risk of untoward side effects in gene therapy.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), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous and exogenous sources, and the balance between NHEJ and HR varies among species, cell types, and cell cycle phases. This review discusses the regulatory factors that control DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated 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 NHEJ or HR, several proteins influence both pathways, including the MRE11/RAD50/NBS1 complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ but 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. HR is considered more accurate for DSB repair, using homologous sequences to prime repair synthesis. However, even with perfect homology, repair can be error-prone. HR is more accurate than NHEJ, but both pathways can lead to genome rearrangements. NHEJ is error-prone but can repair DSBs with complementary overhangs. The choice between NHEJ and HR is regulated by factors such as template availability, cell cycle phase, and the functions of specific cell types. In yeast, HR is more efficient in diploids than haploids, while in higher eukaryotes, HR is more efficient in cells with homologous templates. DNA-PKcs and ATM regulate DSB repair pathway choice, with DNA-PKcs playing a key role in NHEJ and ATM in HR. The regulation of DSB repair pathways is crucial for maintaining genome stability and preventing cancer. Understanding these pathways can lead to clinical benefits in cancer therapy, including more effective chemo- and radiotherapy strategies and improved detection of cancer in its earliest stages. Targeting the proteins that regulate the choice between NHEJ and HR can enhance gene targeting and reduce the risk of untoward side effects in gene therapy.