Claudins are cell adhesion proteins thought to mediate cell–cell contacts at the tight junction. Although a major role of claudins is to control paracellular diffusion, increasing evidence suggests that they may also function in tumor progression. To examine the role of the second extracellular loop in cell adhesion, a small peptide was designed, which mimics a conserved sequence, DFYNP, within specific ‘classic’ claudin subtypes. Using fluorescent indicators with mammary epithelial cells, treatment with both the l- and d-forms of this peptide showed mislocalization of claudin-4 and claudin-3 and activation of caspase-8 and caspase-3, indicating apoptosis. To test specificity, peptides were made both with various end-groups and with glycine substitutions at each of the five residues. Changing end-groups did not influence the activity of the peptide. Amino acid substitutions at F147, Y148, N149, or P150, however, prevented peptide activity. A fluorescent-labeled peptide was shown to associate with the tight junction at 4 °C and cause apoptosis when the cultures were warmed to 37 °C. In conclusion, both the d- and l-forms of a small peptide that mimics a sequence in the second extracellular loop of claudins can target and disrupt claudin proteins in an epithelial monolayer and initiate apoptosis.