Objective: Peripheral nerve injury (PNI) is a significant health problem that is linked to sensory, motor, and autonomic deficits. This pathological condition leads to a reduced quality of life in most affected individuals. Schwann cells (SCs) play a crucial role in the repair of PNI. Effective agents that promote SC activation may facilitate and accelerate peripheral nerve repair. Thymoquinone (TQ), a bioactive component of Nigella sativa seeds, has an antioxidant, anti-inflammatory, immunomodulatory, and neuroprotective properties. In the present study, the neuroprotective efficacy of TQ was investigated by using a laser microdissection technique in a mouse PNI model. Methods: Single cells were isolated from dorsal root ganglions (DRGs) of 6-8-week-old mice, maintained in defined culture conditions and treated with or without TQ at different concentrations. Axons were cut (axotomy) using a controllable laser microbeam to model axonal injury in vitro. Under fluorescence microscopy, cell viability was evaluated using the fluorescent dyes. The behavior of the cells was continuously monitored with time-lapse video microscopy. Results: TQ significantly increased neuronal survival by promoting the survival and proliferation of SCs and fibroblasts, as well as the migration of SCs. Furthermore, TQ improved the ability to extend neurites of axotomized neurons. The regenerative effect of TQ was dose-dependent suggesting a target specificity. Our studies warrant further preclinical and clinical investigations of TQ as a potential regenerative agent to treat peripheral nerve injuries. Conclusion: TQ exhibits a regenerative potential for the treatment of damaged peripheral nerves.