Irinotecan (Ir) is a potent antitumor chemotherapeutics in clinic and used for the treatment of a various cancers, but the degree of its application is critically limited by toxic side-effects and marked heterogeneities. Nano-formulation of prodrugs, based on “all-in-one” carrier-free self-assemblies offers an effective approach to alter pharmacokinetics and safety profiles of cytotoxic agents. In this study, a novel vitamin E succinate-based formulation of Ir (VES-Ir) combined with nanoscaled characteristics and synergistic combination was constructed through esterification. The conjugation makes amphiphilic VES-Ir prodrug self-assemble into nanoparticles with a fine diameter (VES-Ir NPs, 75.4 nm) of spherical morphology. Furthermore, VES-Ir NPs with a 1:1 drug-to-drug ratio was demonstrated to possess respectable physiological stability within 72 h test, while can react to pH/esterase-sensitive drug release in lysosomes internalized into tumor cells, potentially highlighting their alleviating side effects. Compared with single and mixture drugs administration, the nanoformulated VES-Ir NPs codelivered both VES and Ir with different anticancer mechanisms to induce the highest suppress proliferation of MCF-7 (IC50 0.18 μM) and A549 (IC50 0.29 μM) cells in a synergistic way (CI < 1). More importantly, the formulating nanoparticulate Ir is to significantly enhance its bioavailability in vivo with long retention time in bloodstream and thereby, resulting the superior tumor inhibitory rate (TIR) of 85.2% versus controls. This simple nanoformulation of Ir drug deprived from VES conjugation, together with self-delivery and synergistic property, may provide an effective strategy for multiple chemotherapeutics delivery to treat cancers or other diseases.