Objective To evaluate the role of Toll-like receptors (TLR) in host responses to lipopolysaccharide (LPS) by the use of cultured immortalized human comeal fibroblasts (HCF) and to determine whether LPS can induce an antibacterial response in these cells; to evaluate if glucocorticoids can change this response; to investigate the relationship between glucocorticoids and TLR when comeal bacterial infection occurs. Methods The donor's corneal cell was separated, cultured and identified. Cultured HCF were stimulated with LPS (1, 10, 100 ng/ml) from bacteria, and the effect on the expression of TLR was determined by RT-PCR, Real-time polymerase chain reaction (PCR) and immunofluorescence. Cells were also co -cultured with LPS (10 ng/ml) and hydrocortisone (1, 10, 100 μg/ml) to determine whether hydrocortisone modulates the transcription of TLR. Results Incubation of HCF with LPS (1,10,100 ng/ml) up-regulated the expression of all TLR mRNA (TLR2 and TLR4 most noticeably), and when LPS was 10 ng/ml, the expression of TLR mRNA was the highest. Immunofluorescence staining confirmed that expression of TLR2 and TLR4 was up -regulated in response to LPS. This up -regulation was inhibited by co -treatment with hydrocortisone, the expression of TLR mRNA decreased. Conclusion HCF is involved in the corneal immune response to LPS. TLR2 and TLR4 may play a crucial signaling role in response to LPS in HCF. Glucocorticoids can reduce the expression of the TLR on the corneal stroma and thus may reduce the resistance to LPS in the cornea. These findings may provide crucial information for understanding the immune mechanisms of bacterial keratitis and promote the design of new immune therapeutic approaches to bacterial keratitis.