The sclera as well as the cornea forms the principal part of the outer fibrous coat of the eye, with a primary function of protecting the intraocular contents and maintaining the shape of the globe. However, the exact morphometric arrangement of scleral fibroblasts remains unclarified. The aim of this study was to observe the three-dimensional structure of the mouse scleral fibroblasts by focused ion beam/scanning electron microscopy (FIB/SEM). Four eyes from C57BL/6J mice were fixed using a mixture of glutaraldehyde and formaldehyde. The sclera was cut out at the equatorial portion and the posterior pole, and postfixed with potassium ferrocyanide, osmium, thiocarbohydrazide, uranyl acetate and lead aspartate. Specimens were then dehydrated and embedded in an epoxy resin. Serial block face images were obtained using FIB/SEM. Three-dimensional image reconstruction and segmentation of the image stack were created using computer software (Amira v6.0.1, FEI). Scleral fibroblasts were arranged in collagenous layers. The cells frequently showed a cellular junction with the neighboring cells and formed cellular networks. Compared with equatorial fibroblasts, there was a more complicated cellular arrangement of the posterior scleral fibroblasts.