Alpha-mangostin, a natural xanthone mainly extracted from the pericarp of Garcinia mangostana, has been shown to have promising anticancer properties in many types of cancer. However, the therapeutic potential of α-mangostin has been limited so far due to its poor aqueous solubility and low oral bioavailability, which limited its biopharmaceutical applications. Furthermore, α-mangostin failed to specifically reach tumors at a therapeutic concentration due and rapid elimination in vivo. We hypothesized that this drawback could be overcome by loading the drug within a delivery system conjugated to transferrin (Tf), whose receptors are overexpressed on many cancer cells and would enhance the specific delivery of α-mangostin to cancer cells, thereby enhancing its therapeutic efficacy. The objectives of this study were therefore to prepare and characterize transferrin-conjugated lipid-polymer hybrid nanoparticles (LPHN) entrapping α-mangostin, as well as to evaluate their therapeutic efficacy in vitro. We successfully prepared α-mangostin loaded LPHN using a one-step nanoprecipitation method with high drug entrapment efficiency. The conjugation of Tf to the LPHN was achieved by using the thiol-maleimide "click" reaction, leading to an increase in the particle hydrodynamic size of Tf-LPHN compared to that of unconjugated (control) LPHN (Ctrl-LPHN). Both Tf-LPHN and Ctrl-LPHN were bearing negative surface charges. Tf-LPHN and Ctrl-LPHN exhibited a sustained release of α-mangostin at pH 7.4, following an initial burst release, unlike rapid release of drug solution. The entrapment of α-mangostin in the LPHN led to an increase in α-mangostin uptake by cancer cells, and thus improved its antiproliferative activity compared to that observed with the drug solution. In conclusion, α-mangostin entrapped in the Tf-LPHN is therefore a highly promising therapeutic system that should be further optimized as therapeutic tools for cancer treatment.