Background: Red/near-infrared light therapy (R/NIR-LT) has been developed as a treatment for a range of conditions, including injury to the central nervous system (CNS). However, clinical trials have reported variable or sub-optimal outcomes, possibly because there are few optimized treatment protocols for the different target tissues. Moreover, the low absolute, and wavelength dependent, transmission of light by tissues overlying the target site make accurate dosing problematic. Results: In order to optimize light therapy treatment parameters, we adapted a mouse spinal cord organotypic culture model to the rat, and characterized myelination and oxidative stress following a partial transection injury. The ex vivo model allows a more accurate assessment of the relative effect of different illumination wavelengths (adjusted for equal quantal intensity) on the target tissue. Using this model, we assessed oxidative stress following treatment with four different wavelengths of light: 450nm (blue); 510nm (green); 660nm (red) or 860nm (infrared) at three different intensities: 1.93×1016 (low); 3.85×1016 (intermediate) and 7.70×1016 (high) photons/cm2/s. We demonstrate that the most effective of the tested wavelengths to reduce immunoreactivity of the oxidative stress indicator 3-nitrotyrosine (3NT) was 660nm. 860nm also provided beneficial effects at all tested intensities, significantly reducing oxidative stress levels relative to control (p≤0.05). Conclusions: Our results indicate that R/NIR-LT is an effective antioxidant therapy, and indicate that effective wavelengths and ranges of intensities of treatment can be adapted for a variety of CNS injuries and conditions, depending upon the transmission properties of the tissue to be treated.