Heat shock protein A9 (HSPA9), a member of the heat shock protein family, is a putative receptor for Tembusu virus (TMUV). By using Western blot and co-immunoprecipitation assays, E protein domains I and II were identified as the functional domains that facilitate HSPA9 binding. Twenty-five overlapping peptides covering domain I and domain II sequences were synthesized and analyzed by using an HSPA9 binding assay. Two peptides showed the capability of binding to HSPA9. Dot blot assay of truncated peptides indicated that amino acid residues 19 to 22 and 245 to 252 of E protein constitute the minimal motifs required for TMUV binding to HSPA9. Importantly, peptides harboring those two minimal motifs could effectively inhibit TMUV infection. Our results provide insight into TMUV–receptor interaction, thereby creating opportunities for elucidating the mechanism of TMUV entry.