Purpose <p>PARP inhibitors (PARPi) proved effective in Ewing sarcoma cells in preclinical studies. In clinical evaluation, however, the PARPi olaparib failed to elicit substantial responses, suggesting an unknown mechanism of resistance to PARPi in Ewing sarcoma. Since autophagy has been identified as a PARPi resistance mechanism in other tumours, this study aimed at exploring the impact of autophagy on PARPi effectiveness in Ewing sarcoma cells.</p> Methods <p>Effects of the PARPi olaparib and veliparib were assessed by flow cytometry in the Ewing sarcoma cell lines WE-68 and SK-ES-1. Autophagy levels were determined using the autophagosome tracer dye Cyto-ID, and cytotoxic effects were determined by the analysis of cell death and loss of mitochondrial membrane potential. Cyto-ID was used to separate cell populations into subpopulations with low, medium and high autophagy by flow cytometric cell sorting.</p> Results <p>Olaparib and veliparib induced autophagy and cell death in parallel in WE-68 and SK-ES-1 cells. Induction of autophagy and cell death occurred at the same concentrations of both PARPi in both cell lines. Analysis of cells sorted according to autophagy levels revealed a clear association between PARPi effectiveness and autophagy level. The subpopulation of Ewing sarcoma cells with high autophagy responded significantly less to PARPi with cell death than the subpopulation with low autophagy.</p> Conclusion <p>This study demonstrates that autophagy affects the anticancer activity of PARPi in Ewing sarcoma cells.</p>