BACKGROUND: Reactive oxygen species (ROS) such as superoxide radicals, hydrogen peroxide, nitric oxide, and nitroperoxide, cause oxidative stress which interferes with normal cell functioning, resulting in cell damage. It is reported to be associated with chronic pain, especially neuropathic pain, and inflammatory pain. ROS is also closely related to central sensitization. Therefore, this study was designed to explore the effects of Phenyl N-tert-butylnitrone (PBN), an ROS scavenger, in acute, continuous, and increasing pain caused by central sensitization. METHODS: Male Sprague-Dawley rats were divided into 2 groups, an intraperitoneal group (IP) and an intrathecal group (IT), and once again divided into an experimental group and a control group. The experimental group was injected with Phenyl N-tert-butylnitrone (PBN), a free radical scavenger, either intraperitoneally or intrathecally. After inducing pain by injecting formalin into the hind paw, pain behaviors were measured. Lumbar enlargement immmunohistochemistry was performed to assess nitrotyrosine, an oxidative stress marker, to identify the degree of protein nitration. RESULTS: Both experimental groups of IP and IT showed statistically significant decreases in the number of flinches compared to the control group in phase 1 and 2. Immunohistochemical evaluation in the control group revealed an increase in nitrated proteins in the gray matter of the lumbar spinal cord, but a significant decrease in nitrated proteins in the gray matter of lumbar spinal cord of the experimental group. CONCLUSIONS: Intraperitoneal and intrathecal administration of PBN decreases analgesic behaviors, allowing us to believe that ROS is mainly responsible for acute pain and central sensitization.