Background and objective: The aim of study was to investigate the electron microscopic changes in the medulla of the spinal cord that occur with intrathecal midazolam administration. Methods: Twenty-eight albino rabbits of New Zealand type were randomized into two groups. Following anaesthesia, 16 rabbits were given 300 μg of midazolam (Group M) and 12 rabbits were given 0.3 mL of normal saline solution (Group C) intrathecally. Eight rabbits from Group M (Group M1) and 6 rabbits from Group C (Group C1) were sacrificed 24 h after the anaesthesia and 8 rabbits from Group M (Group M2) and 6 rabbits from Group C (Group C2) were sacrificed 6 days after the anaesthesia. The lumbosacral portion was removed by laminectomy and thin sections were examined microscopically. Results: Severe separation in myelin lamella of the large axons, honeycomb appearance, slight separation in myelin lamella of small to moderately large axons, degenerate vacuoles in the cytoplasm and nuclear membrane irregularity were observed in neurons of Groups M1 and M2. Myelin lamella and nuclear membranes were found to be regular, vacuoles and oedema were observed in the neurons in the Groups C1 and C2. Conclusion: Midazolam administered at single dose by the intrathecal route may have neurotoxic effects on the neurons and myelinated axons at 24 h and 6 days following administration.