In neuroblastoma (NB), MYCN genomic amplification and segmental chromosomal alterations including 1p or 11q loss of heterozygocity and/or 17q gain are associated with progression and poor clinical outcome. Segmental alterations are the strongest predictor of relapse and result from unbalanced translocations attributable to erroneous repair of chromosomal breaks. Although sequence analysis of affected genomic regions suggests these errors arise by non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB), abnormalities in NHEJ have not been implicated in NB pathogenesis. On this basis, the hypothesis that an error-prone mechanism of NHEJ is critical for NB cell survival was tested. Plasmid-based DSB repair assays demonstrated efficient NHEJ activity in human NB cells with repair products that were error-prone relative to non-transformed cells. NB cells derived from tumorigenic neuroblastic phenotypes had differential DNA repair protein expression patterns compared to non-tumorigenic cells. Tumorigenic NB cells were deficient in DNA ligase IV (Lig4) and Artemis (DCLRE1C), mediators of canonical NHEJ. Conversely, enzymes required for an error-prone alternative NHEJ pathway (alt-NHEJ), DNA Ligase IIIα (Lig3), DNA Ligase I (Lig1), and poly (ADP-ribose) polymerase-1 protein (PARP1) were upregulated. Inhibition of Lig3 and Lig1 led to DSB accumulation and cell death, linking alt-NHEJ to cell survival in NB. NB cells demonstrated sensitivity to PARP1 inhibition (PARPi) that paralleled PARP1 expression. In a dataset of human NB patient tumors, overexpression of genes encoding alt-NHEJ proteins associated with poor survival. Implications: These findings provide insight into DNA repair fidelity in NB and identify components of the alt-NHEJ pathway as promising therapeutic targets.