Hydrogen peroxide (H2O2) and free radicals cause oxidative stress, which induces cellular injuries, metabolic dysfunction, and even cell death in various clinical abnormalities. Fullerene (C-60) is critical for scavenging oxygen free radicals originated from cell metabolism, and reduced glutathione (GSH) is another important endogenous antioxidant. In this study, a novel water-soluble reduced glutathione fullerene derivative (C-60-GSH) was successfully synthesized, and its beneficial roles in protecting against H2O2-induced oxidative stress and apoptosis in cultured HEK 293T cells were investigated. Fourier Transform infrared spectroscopy and H-1 nuclear magnetic resonance were used to confirm the chemical structure of C-60-GSH. Our results demonstrated that C-60-GSH prevented the reactive oxygen species (ROS)-mediated cell damage. Additionally, C-60-GSH pretreatment significantly attenuated H2O2-induced superoxide dismutase (SOD) consumption and malondialdehyde (MDA) elevation. Furthermore, C-60-GSH inhibited intracellular calcium mobilization, and subsequent cell apoptosis via bcl-2/bax-caspase-3 signaling pathway induced by H2O2 stimulation in HEK 293T cells. Importantly, these protective effects of C-60-GSH were superior to those of GSH. In conclusion, these results suggested that C-60-GSH has potential to protect against H2O2-induced cell apoptosis by scavenging free radicals and maintaining intracellular calcium homeostasis without evident toxicity.