Aspergillus fumigatus triggers inflammatory responses through Toll-like receptors (TLRs), particularly TLR2 and TLR4. In this study, we tested the hypothesis that lipopolysaccharide (LPS), a ligand of TLR4, can induce tolerance of A. fumigatus hyphae in telomerase-immortalized human stroma fibroblasts (THSFs). The THSFs were pretreated with low-dose LPS for various times and then challenged with A. fumigatus hyphae. We observed that pretreatment of THSFs with low-dose LPS resulted in diminished production of cytokines IL-8 and IL-6 and elevated expression of antimicrobial peptides, such as CC chemokine-ligand 20 (CCL20) and thymosin β4 (Tβ4), upon subsequent A. fumigatus challenge. Furthermore, LPS pretreatment also resulted in suppression of polymorphonuclear leukocyte (PMN) migration. Our results suggested that THSFs pretreated with LPS develop a state of A. fumigatus hyphae tolerance. This may induce protective mechanisms during fungal keratitis to prevent an excessive inflammatory response and to control infection of the cornea.