There is currently no effective targeted therapeutic strategy for the treatment of central nervous system acute lymphoblastic leukemia (CNS-ALL). Integrin α6 is considered a potential target for CNS-ALL diagnosis and therapy because of its role in promoting CNS-ALL disease progression. The targeted peptide _D(RWYD) (abbreviated RD), with nanomolar affinity to integrin α6 was identified by peptide scanning techniques such as alanine scanning, truncation, and D-substitution. Herein, we developed a therapeutic nanoparticle based on the α6-targeted peptide for treating CNS-ALL. The self-assembled proapoptotic nanopeptide _D(RWYD)–_D(KLAKLAK)₂–G_D(FFY) (abbreviated RD–KLA–Gffy) contains the α6-targeted peptide RD, the well-known proapoptotic peptide _D(KLAKLAK)₂ (abbreviated KLA) and the self-assembling tetrapeptide G_D(FFY) (abbreviated Gffy). The functional mechanism of RD–KLA–Gffy is clarified using different experiments. Our results demonstrate that RD–KLA–Gffy is highly enriched in CNS-ALL lesions and induces tumor cell apoptosis, thus reducing CNS-ALL disease burden and prolonging the survival of CNS-ALL mice without obvious toxicity. Moreover, the combined use of RD–KLA–Gffy and methotrexate (MTX) shows a potent antitumor effect in treating CNS-ALL, indicating that RD–KLA–Gffy plays an important role in suppressing CNS-ALL progression either as a single agent or in combination with MTX, which shows promise for application in CNS-ALL therapy.