Despite intensive treatment regimens, high-risk and late-stage neuroblastoma tends to have a poor survival outcome. Overexpression of the apoptotic regulator, X-linked inhibitor of apoptosis protein (XIAP), has been associated with chemotherapy resistance in several cancers including neuroblastoma. Here, we report preclinical evidence that XIAP offers an effective therapeutic target in neuroblastoma. Human and murine neuroblastoma cells were treated with the Smac mimetic LBW242 alone or in combination with cytotoxic drugs used clinically to treat neuroblastoma. Expression of XIAP protein, but not mRNA, was highly increased in neuroblastoma cells compared to healthy adrenal gland tissue, consistent with a posttranscriptional regulation of XIAP expression. Treatment with LBW242 sensitized human and murine neuroblastoma cells to chemotherapy-induced apoptosis, which was mediated by activation of both the intrinsic and extrinsic apoptosis pathways. Although Smac mimetics have been reported to stimulate TNF-α-induced apoptosis by degradation of cellular IAP (cIAP)-1/2, we found that LBW242-mediated sensitization in neuroblastoma cells occurred in a TNF-α-independent manner, despite induction of cIAP-1/2 degradation and TNF-α expression. Together, our findings show that XIAP targeting sensitizes neuroblastoma to chemotherapy-induced apoptosis, suggesting a novel therapeutic approach to treat this childhood malignancy.