Neuroblastoma (NB) is the most common extracranial malignant solid tumor in children which contributes to more than 15% of all pediatric cancer-related deaths. Treatment failure in high risk neuroblastoma is largely due to development of chemoresistance. Effective treatments for resistant and recurrent disease remain to be identified. We collected mRNA from six high risk neuroblastoma tumors both at diagnosis and after induction chemotherapy, using laser capture microdissection to obtain a pure population of neuroblasts. Gene expression changes associated with chemotherapy resistance were determined with the aid of the Connectivity Map bioinformatics platform. The analysis identified several therapeutic agents that were predicted to reverse the transcriptome changes associated with chemotherapy resistance. One of these agents, the EWS-FLI1 and RNA helicase A interaction inhibitor YK-4-279, was studied in further detail. Using a panel of neuroblastoma cell lines, including SK-N-AS, SH-SY5Y, IMR-32, CHLA-255, NGP, and NB-19 cells, we found that YK-4-279 had cytotoxic effects on neuroblastoma cells. YK-4-279 also had an inhibitory effect on anchorage-independent growth of neuroblastoma tumor cells and induced cell apoptosis of these cells in vitro. YK-4-279 could enhance Dox-induced cytotoxic effect and cell apoptosis. Moreover, YK-4-279 was able to overcome the established chemoresistance in LA-N-6 neuroblastoma cells. Using an orthotopic neuroblastoma mouse model, we found that YK-4-279 induced apoptosis in tumor cells through PARP and Caspase3 cleavage. Taken together, our results indicate that YK-4-279 might be a promising agent for treatment of refractory neuroblastoma. Furthermore, these results support the general validity of therapeutic strategies that target the chemotherapy-resistance phenotype in malignant tumors.