Abstract: Melampomagnolide B (MMB) is one of the parthenolide (PTL) derivatives with high anticancer activity to various tumors. However, its application in the clinic is limited due to its poor water solubility. To overcome this problem, an amphiphilic prodrug is synthesized from carboxyl polyethylene glycol monomethyl ether (mPEG₁₀-COOH) and MMB through an esterification reaction. The chemical structure of mPEG₁₀-MMB is confirmed by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS). The amphiphilic prodrug mPEG₁₀-MMB can self-assemble in water with the critical micelle mass concentration of 7.7 μg/mL and the size/morphology of its assemblies is characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The DLS result indicates that an averaged size of mPEG₁₀-MMB nanoparticles is about 120.3 nm with a narrow distribution. The TEM image exhibits that mPEG₁₀-MMB can self-assemble into spherical nanoparticles with an average diameter of 108.5 nm. Nile red (NR) is used as the fluorescent probe and loaded in mPEG₁₀-MMB prodrug nanoparticles. The flow cytometry and confocal laser scanning microscope (CLSM) are used to evaluate the cell uptake of mPEG₁₀-MMB prodrug nanoparticles. The results demonstrate that mPEG₁₀-MMB prodrug nanoparticles can be internalized by HeLa cells efficiently through the endocytosis mechanism. In vitro cytotoxicity of mPEG₁₀-MMB prodrug nanoparticles is evaluated against HeLa cells and BRL-3A cells by MTT assay. The result demonstrates that mPEG₁₀-MMB prodrug nanoparticles have higher cytotoxicity to cancer cells compared to that of free MMB, but relatively lower cytotoxicity to normal cells.