The present study explored the cellular uptake dynamics, the subcellular location and the internalization mechanisms of gold nanoparticles (GNPs) and glucose-capped GNPs (Glu-GNPs). The cancer radiotherapy-enhancing effects of GNPs were also evaluated. We synthesized the GNPs and Glu-GNPs by the seeding technique. The effects on cellular uptake and the radiosensitizing effect induced by GNPs and Glu-GNPs at lower doses were investigated using two human cancer cell lines (HeLa and MCF-7). The intracellular location of the nanoparticles was analyzed by transmission electron microscopy (TEM). Analysis of cellular apoptosis following GNP-based radiotherapy was performed by flow cytometry and TUNEL assay. Cancer cells took up more Glu-GNPs than naked GNPs and the uptake curve showed size- and cell-dependent uptake. GNPs were mainly located in the cytoplasm and endocytosis is the mechanism behind the internalization of GNPs and Glu-GNPs. Lower doses of GNPs and Glu-GNPs still enhanced the killing effect using X-ray irradiation, although the apoptotic rate was not altered. The results presented in this study provide evidence that Glu-GNPs may have a bright future in tumor-targeted diagnosis and treatment.