<span class="paragraphSection"><div class="boxTitle">Abstract</div>DNA double-strand breaks (DSBs), the most lethal DNA lesions, are repaired primarily by homologous recombination (HR) or nonhomologous end joining (NHEJ). Caffeine is known to inhibit HR by displacing Rad51 from single-stranded DNA, but its impact on NHEJ was unclear. Here, we show that caffeine inhibits NHEJ in a concentration-dependent manner using biochemical and cellular assays. Increased 53BP1 and γ-H2AX foci upon caffeine exposure indicate inhibition of chromosomal NHEJ, leading to accumulation of DSBs. γ-H2AX immunofluorescence, neutral comet, and TUNEL assays revealed persistent DNA breaks and reduced repair. Mechanistically, <span style="font-style: italic;">in silico</span>, biophysical, and biochemical analyses demonstrate that caffeine directly binds to XRCC4, disrupting its interaction with DNA ligase IV and thereby inhibiting repair. Biolayer interferometry confirmed caffeine–XRCC4 binding, with mutation of Thr133 reducing caffeine affinity and impairing XRCC4 recruitment to γ-H2AX–marked DSBs. Disruption of the predicted caffeine interaction site in XRCC4 (T133A) partially restored end joining in the presence of caffeine. Clonogenic survival assays showed decreased survival after caffeine treatment, more prominently in wild-type than in ligase IV–deficient cells. Immunodepletion and reconstitution experiments confirmed that caffeine specifically targets the ligase IV/XRCC4 complex. Thus, caffeine suppresses NHEJ by directly inhibiting ligase IV/XRCC4–mediated DNA end joining.</span>