AIM: To investigate the positive rate and types of cells that express Epstein-Barr virus-encoded small RNAs (EBERs) and to determine the distribution of EBER-expressing cells in idiopathic orbital inflammatory pseudotumor (IOIP) tissues. METHODS: We retrospectively examined 40 archived paraffin specimens from two teaching hospitals in Southern China between January 2007 and January 2015 that were pathologically determined to exhibit IOIP. Eleven concurrent paraffin specimens of thyroid-associated ophthalmopathy (TAO) composed the control group. In situ hybridization was performed to detect EBERs. Immunohistochemistry was employed to detect CD3, CD20, Vimentin, and smooth muscle actin (SMA), and the positive rate, types of positive cells, and distribution and location of EBERs were evaluated. RESULTS: The positive expression rate of EBERs was 47.5% (19/40) in the IOIP group, which was significantly higher than that in the TAO group [0 (0/11), P=0.011]. In the IOIP group, the lymphocyte infiltrative subtype, fibrotic subtype, and mixed subtype exhibited EBER-positive rates of 57.1% (12/21), 12.5% (1/8), and 54.5% (6/11), respectively, and no significant differences were found between these subtypes (P=0.085). Positive signals of EBERs were mainly present in medium-small lymphocytes between or around follicles and in the nuclei of activated immunoblasts (14/19). CONCLUSION: The positive rate, types, and distribution of EBER-expressing cells in IOIP have been documented. These findings are conducive for a better understanding of the underlying mechanisms of Epstein-Barr virus infection in IOIP pathogenesis.