Xanthones are a group of oxygenated heterocyclic compounds with anticancer properties, but poor aqueous solubility and low oral bioavailability hinder their therapeutic application. This study sought to prepare a xanthones extract (81% α-mangostin and 16% γ-mangostin) in polymeric nanoparticles and to investigate its intracellular delivery and cytotoxicity toward colon cancer cells. The nanoparticles were prepared in Eudragit RL100 and Eudragit RS100 by the nanoprecipitation method at drug loading and entrapment efficiency of 20% and >95%, respectively. Freeze-drying of bulk nanoparticle solutions, using glucose or sucrose as cryoprotectants, allowed the collection of nanoparticles at >95% yield. Solubility of the xanthones extract was improved from 0.1 µg/mL to 1250 µg/mL. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) of the freeze-dried final formulation showed the presence of cationic round nanoparticles, with particle size in the range of 32–130 nm. Scanning electron microscopy (SEM) showed the presence of nanospheres, and Fourier transform infrared (FTIR) spectroscopy indicated intermolecular interaction of xanthones with Eudragit polymers. Cellular uptake of nanoparticles was mediated via endocytosis and indicated intracellular delivery of xanthones associated with potent cytotoxicity (median inhibitory concentration26.3±0.22 µg/mL). Presented results suggest that cationic nanoparticles of xanthones may provide a novel oral drug delivery system for chemoprevention or treatment of intestinal and colon tumors.