Rotavirus (RV) VP8* peptide-induced CLDN3 mislocalization supports the hypothesis that CLDN3 negatively regulates viral binding, while the molecular basis of this inhibitory function remains unresolved. To counteract the CLDN3 defense strategies, RV infection indeed disrupts its localization to the plasma membrane. We also found that RV infection could reduce its protein levels in both in vitro and animal models. Knockdown or knockout of CLDN3 effectively promotes RV binding and entry. Further, we found that CLDN3 EC1 loop could interact with the N-terminal domain of VP7 and structural studies reveal a conserved glutamic acid at position 74 (E74) in VP7 as critical for the CLDN3-VP7 interaction. Mechanistically, VP7 is involved in viral attachment. Binding of the CLDN3 EC1 loop to VP7 reduces viral adsorption, whereas the E74K mutation disrupts the CLDN3-VP7 interaction and consequently enhances viral attachment. More importantly, a single E74K mutation enhances viral pathogenicity in vivo, confirming this interaction's biological significance. Our results demonstrate for the first time that the tight junction protein CLDN3 acts as a decoy receptor that specifically counters the VP7-mediated viral attachment. This highlights the antiviral mechanisms utilized by CLDN3.