Purpose: To investigate the pathogenesis of a secondary iris cyst with an immunohistochemical method. Methods: Single observational case report. A pathologic specimen was obtained from a 5-year-old girl who was found to have a secondary iris cyst. She had a history of previous penetrating ocular trauma and subsequent cataract surgery and pupilloplasty. Immunohistochemical staining with cytokeratin (CK) 19 and CK3 was used. Results: After immunohistochemical staining, the inner wall showed positive staining for CK19, which is specific for limbal, peripheral cornea and conjunctival epithelium, and negative staining for CK3, which is specific for corneal epithelium. Conclusions: With the aid of immunohistochemical analysis, a conjunctival epithelial origin was indicated, and pupilloplasty was identified as the causal event of the iris cyst while the possibility of primary iris cyst was ruled out.