To study the effect of an inverted internal limiting membrane (ILM) flap on the retina. Prospective case series. Twenty-nine patients with large (>400 μm) unilateral macular holes underwent surgery using a modified inverted ILM flap technique. The macular ILM was peeled, and a large (2-3 disc diameter) ILM flap was made on the superior side of the hole, and then the flap was inverted on the inferior side. In all patients, multifocal electroretinograms (mfERGs) were recorded from operated eyes and normal fellow eyes. The peak time and amplitude of N1, P1, and N2 in the focal ERG from the upper retina without the ILM flap and those from the lower retina with the ILM flap were evaluated. In 14 patients, microperimetry was also performed in both eyes, and the averaged sensitivity was measured from the upper and lower areas. The peak times of P1 and N2 from the upper and lower retina were significantly longer in operated eyes than in the fellow eyes (P1 upper and lower: P < 0.04, N2 upper: P < 0.01, and N2 lower: P < 0.04), although we could not identify a significant difference in peak time and amplitude of N1, P1, and N2 between the upper retina and lower retina in both eyes (fellow eye-N1 amplitude: P > 0.2, N1 peak time: P > 0.5, P1 amplitude: P>0.9, P1 peak time: P > 0.4, N2 amplitude: P > 0.9, N2 peak time: P > 0.9; operated eye-N1 amplitude: P>0.8, N1 peak time: P > 0.4, P1 amplitude: P > 0.6, P1 peak time: P > 0.4, N2 amplitude: P > 0.9, N2 peak time: P > 0.9). We could not observe a significant difference in sensitivity between the upper and lower retinas in both eyes (fellow eye: P= 0.28, operated eye: P= 0.66). The results of this study revealed no significant difference between the upper retina without the ILM flap and the lower retina with the ILM flap, suggesting that an inverted ILM flap has little effect on retinal function.