A novel type of stress proteins has been identified in mammals to defend environmental stresses and maintain tissue integrity. Cornulin (CRNN) that contains S100 EF-hand Ca²⁺-binding motif is a stress protein highly expressed in the human esophageal squamous epithelial cells. It is downregulated in esophageal squamous cell carcinoma and functions as a modifier of deoxycholic acid (DCA) mediated cell injury. The S100 domain may be central to the function of CRNN. To further characterize the S100 domain of CRNN, the S100 domain in Escherichia coli, was cloned, expressed, purified and demonstrated that it was properly folded and suitable for biochemical and biophysical studies. More importantly, by nuclear magnetic resonance, gel-filtration, analytical ultracentrifugation, electrospray ionization-mass spectrometry, and Cross-linking analyses, a Ca²⁺-dependent multimeric property of S100 domain was identified. Furthermore, in response to DCA and ethanol challenge, the multimers have stronger protective effects on cells than dimers do. These data indicate that the S100 domain is a key domain in CRNN, which functions as a survival factor through multimerization. This work helps to further understand the feature of S100 domain and its association with cell injury.