7-ethyl-10-hydroxy-camptothecin (SN38) is a broad-spectrum antitumor agent. Unfortunately, the application of SN38 is greatly limited by its poor solubility. In response, irinotecan, the hydrophilic derived prodrug of SN38, has been developed into commercial formulation Campto® for the treatment of colorectal cancer. However, only 1% to 0.1% of irinotecan could be transferred to active SN38 in vivo, leading to the unsatisfactory antitumor activity in clinic. Herein, we report a smart-responsive SN38 prodrug nanoassembly for efficient cancer therapy. First, SN38 was conjugated with an endogenous lipid cholesterol (CST) via a redox dual-responsive disulfide bond (SN38-SS-CST). The prodrug could self-assemble into uniform prodrug nanoassemblies with good colloidal stability and ultrahigh drug loading. Under the redox environments of tumor cells, SN38-SS-CST NPs could sufficiently release SN38 while keep intact in normal tissues. Finally, SN38-SS-CST NPs potently defeated the growth of colon cancer without systemic toxicity, promising a translational chemotherapeutic nanomedicine.