Although the cure rate of newly diagnosed acute lymphoblastic leukemia (ALL) has improved over the past four decades, the outcome for patients who relapse remains poor. New therapies are needed for these patients. Our previous global gene expression analysis in a series of paired diagnosis-relapse pediatric patient samples revealed that the antiapoptotic gene survivin was consistently upregulated upon disease relapse. In this study, we demonstrate a link between survivin expression and drug resistance and test the efficacy of a novel antisense agent in promoting apoptosis when combined with chemotherapy. Gene-silencing experiments targeting survivin mRNA using either short-hairpin RNA (shRNA) or a locked antisense oligonucleotide (LNA-ON) specifically reduced gene expression and induced apoptosis in leukemia cell lines. When used in combination with chemotherapy, the survivin shRNA and LNA-ON potentiated the chemotherapeutic antileukemia effect. Moreover, in a mouse primary xenograft model of relapse ALL, the survivin LNA-ON decreased survivin expression in a subset of animals, and produced a statistically significant decrease in tumor progression. Taken together, these findings suggest that targeting endogenous levels of survivin mRNA by LNA-ON methods may augment the response to standard chemotherapy by sensitizing otherwise resistant tumor cells to chemotherapy.