The synthesis of intrinsically radiolabeled nanoparticles has shown tremendous prospective in offering an easier, faster, stable, and more specific radiolabeling technique for the development of advanced radionanomedicine agents for cancer treatment. In this study, a facile one-pot synthesis protocol for the preparation of cyclic arginine−glycine−aspartate (RGD) conjugated and intrinsically radiolabeled 199 Au (t 1/2 = 3.14 d; β 1 = 462 keV, 6.0%; β 2 = 296 keV, 71.6%; β 3 = 250 keV, 22.4%; γ = 159 keV, 37%) nanoparticles has been developed for targeting integrin α v β 3 receptors for potential use in neoadjuvant brachytherapy. The nanoparticles synthesized by this method were characterized by numerous analytical methods to determine the identity, particle size, in vitro stability, biocompatibility, and amenability for clinical use. Large-scale synthesis of intrinsically radiolabeled 199 Au nanoparticles could be achieved with excellent yield, and they met all the requirements for clinical use. The biological efficacy of the intrinsically radiolabeled 199 Au nanoparticles was confirmed by biodistribution studies in C57BL/6 mice having melanoma tumors after intratumoral injection. The results of the biodistribution studies demonstrated high tumor radioactivity concentration of integrin α v β 3 targeted 199 Au nanoparticles which reduced only marginally over the period of 1 week. Intratumoral administration of 5 MBq and 10 MBq doses of radiolabeled nanoparticles resulted in significant regression of tumor growth in melanoma tumor bearing mice. Apparent body weight loss was not observed in all the treated mice. On the basis of the encouraging results acquired in this study, it is envisaged that this approach would be useful toward clinical translation of this innovative class of radionanomedicine agents for neoadjuvant brachytherapy of selected cancer patients.