Prodrug, in which the inactive parent drug with good bioavailability is metabolized into an active drug in the body, is one of the main strategies to target the disease site to improve the drug efficiency and reduce the adverse effects of chemotherapy. Because of the good capability of chemical modification, zein, a plant derived protein, and drugs can be conjugated through environmentally sensitive links to form prodrugs capable of triggered drug release. In this study, a novel prodrug was synthesized using paclitaxel (PTX), zein, and a disulfide linker, and nanoparticles were formed by self-assembly of the prodrug. An effective in vitro triggered release, 80-90% in 5 min, of the prodrug based nanoparticles (zein-S-S-PTX_NP) was successfully approached. The cytotoxicity of zein-S-S-PTX_NP as well as the zein encapsulation of PTX (zein_PTX_NP) and pure PTX on HeLa cells and NIH/3T3 fibroblast cells was tested using MTS assay. It showed that, after the treatment of zein-S-S-PTX_NP at the equivalent PTX concentrations of 0.1, 0.5, 1, and 5 μg/mL, respectively, zein-S-S-PTX_NP had zero damage to normal cells but a similar cytotoxicity to cancer cells as pure PTX. In the animal study, the tumor was 50% of the original size after the treatment of zein-S-S-PTX_NP for 9 days with 3 doses. This study suggested that the novel prodrug based nanoparticle zein-S-S-PTX_NP could be a promising approach in chemotherapy with targeted delivery, improved efficacy, and reduced side effects.