The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), is considered an important regulator of steroidogenesis and a potential therapeutic target in neurological disorders. Previous evidence suggests that TSPO ligands can protect cells during injury and prevent apoptosis in central nervous system (CNS) cells. However, its actions on astrocytic cells under metabolic injury are not well understood. In this study, we explored whether 4′-chlorodiazepam (Ro5–4864), a TSPO ligand, might protect astrocyte mitochondria under glucose deprivation. Our results showed that 4′-chlorodiazepam preserved cell viability and reduced nuclear fragmentation in glucose-deprived cells. These effects were accompanied by a reduced production of free radicals and maintenance of mitochondrial functions in cells treated with 4′-chlorodiazepam. Finally, our findings suggest that TSPO might be involved in reducing oxidative stress by preserving mitochondrial functions in astrocytic cells exposed to glucose withdrawal.