Acute lymphoblastic leukemia (ALL) is a common malignant disease and a major cause of mortality due to recurrent disease. Immunotherapy is a promising strategy for eradicating minimal residual disease and thus preventing the relapse of leukemia. Apart from stem cell transplantation, CpG oligodeoxynucleotides (ODNs) are excellent candidates for the immunotherapy of leukemia. However, the number of usable CpG ODNs is limited. In this study, we tested a panel of CpG ODNs and obtained three CpG ODN sequences with strong immunostimulatory activity by comparing their capacity to activate lymphocytes. The data revealed that the flanking bases, the spacing of individual CpG motifs and polyguanosine ends, contribute to the immunostimulatory activity of a CpG ODN. In the immunotherapy of murine leukemia with the novel ODN as the adjuvant, we found that CpG Seqs 14 and 19 were effective in the treatment of a leukemia model by prolonging survival span, augmenting natural killer cell and CTL cytotoxicity, as well as increasing the number of long-term survivors. The ability of CpG ODN to induce both strong innate and adaptive anti-leukemic immune activity could render it an appropriate agent for therapeutic applications in acute leukemia. This study demonstrates the feasibility of active immunotherapy with CpG ODNs in patients with acute leukemia and thus represents a potential alternative therapeutic strategy for eradicating residual disease which is resistant to conventional cytoreductive treatment.