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We recently identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within the fibrin βN-domains and the third and fifth extracellular domains (EC3 and EC5) of N-cadherin. We also hypothesized that the His16 and Arg17 residues of the βN-domains and the (Asp/Glu)-X-(Asp/Glu) motifs present in the EC3 and EC5 domains may play roles in the interaction between fibrin and N-cadherin. The primary objectives of this study were to test these hypotheses and to further clarify the structural basis for this interaction. To test our hypotheses, we first mutated His16 and Arg17 in the recombinant (β15–66)2fragment, which mimics the dimeric arrangement of the βN-domains in fibrin, using site-directed mutagenesis. The results revealed that the mutations of both His16 and Arg17 are critical for the interaction. Next, we mutated Asp/Glu residues in the three (Asp/Glu)-X-(Asp/Glu) motifs, M1 (Asp-Phe-Glu), M2 (Glu-Ala-Glu), and M3 (Asp-Tyr-Asp), of the fibrin-binding N-cad(3–5) fragment of N-cadherin. The results showed that Asp292 and Glu294 of M1, and Asp468 and Asp470 of M3, are critical for the interaction. Our molecular modeling of the 3D structure of the EC3-EC4-EC5 domains revealed that these residues are located at the interfaces of EC3-EC4 and EC4-EC5 and that some may also be involved in calcium binding. In conclusion, our study identified amino acid residues in the fibrin βN-domains and the EC3 and EC5 domains of N-cadherin that are critical for the interaction of fibrin with N-cadherin and localized the fibrin-binding residues in the 3D structure of N-cadherin.