Purpose: This study was performed in order to characterize the psychometric properties of the National Eye Institute visual functioning questionnaire in Iranians. Methods: After forward and backward translation, examination of the translation quality and a pilot test, 80 patients with various chronic ophthalmic diseases and 30 healthy individuals completed the questionnaire. Internal consistency (IC) was measured using Cronbach's alpha coefficient and reproducibility was evaluated using the intraclass correlation coefficient (ICC) obtained through test-retests. Regarding construct validity, convergent, discriminant and known group comparison validities were evaluated. The standardized response mean index was used to assess responsiveness and sensitivity of the instrument to changes. Results: Cronbach‟s alpha was above 0.7 for all of the subscales except for that of “driving”, which had a value of 0.68. The ICC in all subscales was above 0.7. All items had correlations higher than 0.4 with their original subscales. About 70% of the items were correlated with their own subscale more than other subscales. Known group comparison showed that the healthy group scored significantly higher than the patients in all subscales and the composite score (P<0.001). Standardized response means ranged from 0.61 to 2.42, and was 1.19 in general health (GH), indicating the sensitivity of the instrument to changes. Conclusion: The Persian version of the National Eye Institute visual functioning questionnaire 25 was valid, reliable, responsive to changes and could evaluate the results of therapeutic ophthalmic interventions and quality of life (QoL) of the Iranian patients.