AIM: To assess the effect of experimentally induced anisometropia on binocularity in normal adults with glasses-free three-dimensional (3D) technique. METHODS: Totally 54 healthy medical students with normal binocularity in the cross-sectional study were enrolled. Anisometropia was induced by placing trail lenses over the right eye, in 0.5 D steps including lenses of -0.5, -1, -1.5, -2, -2.5 D (hyperopic anisometropia) and lenses of +0.5, +1, +1.5, +2, +2.5 D (myopic anisometropia). The glasses-free 3D technique was used to evaluated not only fine stereopsis, but also coarse stereopsis, dynamic stereopsis, foveal suppression, and peripheral suppression in these subjects. One-way analysis of variance was used to compare quantitative data such as fine stereopsis, coarse stereopsis. Pearson’s Chi-square test was performed to compare categorical data such as dynamic stereopsis, foveal suppression and peripheral suppression. RESULTS: The subjects showed a statistically significant decline in fine stereopsis, coarse stereopsis, and dynamic stereopsis with increasing levels of anisometropia (P<0.001). Binocularity was affected when induced anisometropia was more than 1 D (P<0.05). Foveal suppression and peripheral suppression were evident and increased in proportion to anisometropia (P<0.001). CONCLUSION: The relatively low degrees of anisometropia may have a potentially significant effect on high-grade binocular interaction. The mechanisms underlying the defect of binocularity seem to involve not only foveal suppression, but also peripheral suppression.