Alternative end-joining (alt-EJ) is a DNA end resection-dependent, error-prone pathway utilized by vertebrate cells to repair DNA double-strand breaks (DSBs), but its engagement is linked to chromosomal translocations and genomic instability. Here, we report that when proliferating cells are exposed to ionizing radiation, treatment with nucleoside analogs (NAs) causes strong radiosensitization by increasing engagement of alt-EJ, while at the same time suppressing homologous recombination (HR) in S- and G₂phase cells. This NA-mediated pathway shift may reflect a passive compensatory engagement of alt-EJ following HR suppression that is specific for S- and G₂-phase cells, and/or the direct activation of alt-EJ throughout the cell cycle. To distinguish between these possibilities, we utilize here a cell culture model that exploits genetic and cell cycle-dependent inactivation of DSB repair pathways, to exclusively study alt-EJ and its modulation by NAs in murine and human cell lines. To this end, we allow LIG4^–/–-deficient cells to accumulate in G₁/G₀ phase by transfer to serum-deprived media and obtain cells deficient in c-NHEJ owing to the genetic LIG4 knockout, deficient in HR owing to the absence of S- or G₂-phase cells, and compromised in their ability to carry out alt-EJ owing to their accumulation in G₀. We find that in these cells irradiation and treatment with the NA, β-arabinofuranosyladenine (araA), and to a lesser degree with other NAs, promptly activates suppressed alt-EJ that now functions at levels approximating those of c-NHEJ in wild-type cells. Results at high dose (20 Gy) generated using pulsed-field gel electrophoresis (PFGE) are corroborated by results at low dose (1 Gy) generated by scoring 53BP1 foci. Strikingly, araA treatment activates a normally undetectable DNA-end-resection at DSBs, which requires ATR activity, but proceeds unimpeded after CtIP knockdown. Treatment with araA increases the formation of chromosomal aberrations and enhances radiation-induced cell killing. The results support direct stimulation of resection by NAs and alt-EJ as a mechanism of their documented radiosensitizing potential. We propose that this stimulation also occurs in repair-proficient cells and that it occurs throughout the cell cycle. It may therefore be harnessed to develop protocols combining NAs with radiation to treat human cancer.