High rates of relapsed and refractory diffuse large B-cell lymphoma (DLBCL) patients and life-threatening side effects associated with immunochemotherapy make an urgent need to develop new therapies for DLBCL patients. Immunotoxins seem very potent anticancer therapies but their use is limited because of their high toxicity. Accordingly, the self-assembling polypeptidic nanoparticle, T22-DITOX-H6, incorporating the diphtheria toxin and targeted to CXCR4 receptor, which is overexpressed in DLBCL cells, could offer a new strategy to selectively eliminate CXCR4 ⁺ DLBCL cells without adverse effects. In these terms, our work demonstrated that T22-DITOX-H6 showed high specific cytotoxicity towards CXCR4 ⁺ DLBCL cells at the low nanomolar range, which was dependent on caspase-3 cleavage, PARP activation and an increase of cells in early/late apoptosis. Repeated nanoparticle administration induced antineoplastic effect, in vivo and ex vivo , in a disseminated immunocompromised mouse model generated by intravenous injection of human luminescent CXCR4 ⁺ DLBCL cells. Moreover, T22-DITOX-H6 inhibited tumor growth in a subcutaneous immunocompetent mouse model bearing mouse CXCR4 ⁺ lymphoma cells in the absence of alterations in the hemogram, liver or kidney injury markers or on-target or off-target organ histology. Thus, T22-DITOX-H6 demonstrates a selective cytotoxicity towards CXCR4 ⁺ DLBCL cells without the induction of toxicity in non-lymphoma infiltrated organs nor hematologic toxicity.