Objective To study the mechanism of Toll-like receptor 7 (TLR7) signal transduction by identifying the critical amino acid in TLR7 Toll/Interleukin-1 receptor (TIR) domain. Methods We carried out a series of truncating mutagenesis and site-direct mutagenesis to obtain TLR7 mutants. These TLR7 mutants were transfected into HEK293T cells. NF-κB signal pathway activation was determined by the reporter gene assay. Immunoprecipitation and immunoblotting were employed to determine the binding between TLR7 and MyD88. Results We identified a RXR signal motif with two arginines at position 1004 and 1006, which plays a significant role in TLR7 signal transduction. We further demonstrated that only the arginine at position 1004 could affect the binding between TLR7 and MyD88. Conclusion We conclude that the arginine at position 1004 of human TLR7 is critical for its binding with MyD88 in signal transduction.