Objectives: Keratoacanthoma is a dome-shaped skin tumor with a centralized keratinous plug, characterized by initial rapid growth and often spontaneous regression. Cytotoxic T-lymphocyte-associated protein 4 or CTLA-4 is an inhibitory receptor, belonging to the CD28 immunoglobulin subfamily, that inhibits T cell activation and mediates regulatory T-cell suppressive activity. The aim of this study was to determine whether there is a difference in CTLA-4 expression in the keratoacanthoma center, at the base and in the healthy lateral resection margin as a control, as well as to determine whether it is dependent on keratoacanthoma stage, sex, and age. Materials and methods: The study included 30 samples of paraffin-embedded tissue, obtained after KA excision. Anti–CTLA-4 rabbit antibody (ab237712, Abcam, UK) and a secondary antibody labeled with fluorescein were used for immunofluorescence staining. The slides were analyzed by fluorescence microscopy. The number of CTLA-4-positive cells was expressed as a percentage of the total number of cells per field of view (400 x). Results: CTLA-4 protein was expressed in the tumor center in 28 (93.33%) cases, at the tumor base in 28 (93.33%) cases, and significantly less on the healthy lateral resection margin, in 17 (56.67%) samples. Statistically significant difference in CTLA-4 expression between the stages of KA was found in the central part of the tumor (P = 0.020). Also, statistically significant difference was found between the group with the proliferative stage and the group without exact stage of keratoacanthoma (P = 0.037), as well as between the group with the proliferative stage and the group with the regression stage of KA (P = 0.022). No statistically significant gender difference was found in CTLA-4 expression in the tumor center (P = 0.546), tumor base (P = 0.151), or at the healthy lateral resection margin (P = 0.914). No statistically significant difference was found between age groups with respect to CTLA-4 expression in the tumor center (P = 0.592), at the tumor base (P = 0.888), and at the healthy resection margin (P = 0.821). Conclusions: CTLA-4 expression is significantly higher in the tumor (center and base) than on the healthy lateral resection margin. The regression stage of keratoacanthoma has a higher expression of CTLA-4 than the proliferative stage. Gender and age were not associated with CTLA-4 expression. To further clarify the role of CTLA-4 in the development and/or in the regression of keratoacanthoma, additional research is needed to determine the phenotype of CTLA-4 positive cells.