AIM: To detect the expression of TLR4 in rats corneal epithelium with pretreatment of Aspergillus. METHODS: Healthy Wistar rats were randomly divided into four groups of A, B, C and D. Group A was the blank control group. Rats corneal epithelium was stimulated by Aspergillus for 4 hours in group B,C and D. Rats corneal epithelium was scraped immediately in the completion of the stimulation in group B. The epithelium was scraped after 96 hours in group C. The epithelium was stimulated again by Aspergillus after 96 hours in group D, and was scraped immediately. Real-time PCR was used to assay the mRNA expression of TLR4 receptor in the epithelial tissue. RESULTS: The expression of TLR4 was significantly increased by the stimulation of Aspergillus, and it became normal after 96 hours. The expression of TLR4 was no significant difference after twice stimulations. CONCLUSION: The expression of TLR4 can be increased by the stimulation of Aspergillus, but the basic expression level has no significant change.