Abstract Toll-like receptor 4 (TLR4) has been demonstrated to play an important role during aseptic inflammation caused by nervous system diseases. The purpose of this study was to explore the underlying mechanism(s) regulating TLR4-mediated signaling and aseptic inflammatory responses following optic nerve injury in mice. We successfully generated an optic nerve crush model in mice in which the optic nerve upregulated TLR4 following injury. The protein expression levels of TLR4, Mac1, MyD88, NF-κB, IL-6 and TNF-α were significantly increased after optic nerve injury. Moreover, the expression levels of TLR4, NF-κB and TNF-α were robust at 2 weeks following injury; however, TRIF protein expression levels were low. In addition, we found that the mRNA transcript levels of MyD88 were higher than TRIF. IL-6 and TNF-α exhibited a statistically significant increase in their expression at 1 day, 1 week, 2 weeks and 3 weeks after optic nerve injury, compared with the normal and sham control groups (p-value <0.05). Therefore, the TLR4-mediated signaling pathway is activated following optic nerve injury in mice. We found that TLR4-MyD88-NF-κB signaling is the main signaling pathway activated in TLR4-mediated inflammation. Our results suggest this pathway may be a main pathway mediating inflammatory responses following optic nerve injury. This may provide insight into novel regenerative targets for the treatment of nerve injury.