Claudins (Cld) are essential constituents of tight junctions. Domain I of Clostridium perfringens enterotoxin (cCPE) binds to the second extracellular loop (ECL2) of a subset of claudins, e.g. Cld3/4 and influences tight junction formation. We aimed to identify interacting interfaces and to alter claudin specificity of cCPE. Mutagenesis, binding assays, and molecular modeling were performed. Mutation-guided ECL2 docking of Cld3/4 onto the crystal structure of cCPE revealed a common orientation of the proposed ECL2 helix-turn-helix motif in the binding cavity of cCPE: residues Leu(150)/Leu(151) of Cld3/4 bind similarly to a hydrophobic pit formed by Tyr(306), Tyr(310), and Tyr(312) of cCPE, and Pro(152)/Ala(153) of Cld3/4 is proposed to bind to a second pit close to Leu(223), Leu(254), and Leu(315). However, sequence variation in ECL2 of these claudins is likely responsible for slightly different conformation in the turn region, which is in line with different cCPE interaction modes of Cld3 and Cld4. Substitutions of other so far not characterized cCPE residues lining the pocket revealed two spatially separated groups of residues (Leu(223), Asp(225), and Arg(227) and Leu(254), lle(258), and Asp(284)), which are involved in binding to Cld3 and Cld4, albeit differently. Involvement of Asn(148) of Cld3 in cCPE binding was confirmed, whereas no evidence for involvement of Lys(156) or Arg(157) was found. We show structure-based alteration of cCPE generating claudin binders, which interact subtype-specific preferentially either with Cld3 or with Cld4. The obtained mutants and mechanistic insights will advance the design of cCPE-based modulators to target specific claudin subtypes related either to paracellular barriers that impede drug delivery or to tumors.