Thymoquinone (TQ) exhibits a wide spectrum of biological activities. Most studies on the neurotoxic action of TQ have been carried out in cancer cell lines. Here, we studied the toxic effect of TQ in primary neuronal cultures in vitro. Incubation with 0.04-0.05 mM TQ for 24 h induced the death of cultured cerebellar granule neurons (CGNs) in a dose-dependent manner. Neuronal death was preceded by an increase in the reactive oxygen species (ROS) generation, as demonstrated using CellROX Green and MitoSOX Red. Confocal and electron microscopy showed that incubation with 0.05 mM TQ for 5 h induced changes in the intracellular localization of mitochondria and mitochondria hypertrophy and cell swelling. The antioxidant N-acetyl-L-cysteine (2 mM) protected CGNs from the toxic action of TQ. Taken together, these facts suggest that TQ is toxic for normal neurons, while ROS-induced changes in the mitochondria can be one of the major causes of the TQ-induced neuronal damage and death.