PURPOSE. Dermatopontin (DPT) is an abundant component of the stromal extracellular matrix; however, its function in the cornea is poorly understood. This study was conducted to determine whether DPT has a direct role in corneal matrix organization by investigating the ultrastructure of Dpt-null (Dpt–/–) mouse corneas. METHODS. Conventional light microscopy was used to compare the corneal thickness of Dpt–/– mice with that of the wild type. Collagen fibril distribution was studied using transmission electron microscopy and the datasets analyzed using image analysis software to determine fibrillar volume, fibril diameter, and spacing. RESULTS. Light microscopy demonstrated that Dpt–/– corneas in 2-month-old mice showed a 24% reduction in average stromal thickness compared with wild type (P < 0.001). The epithelium and Descemet’s membrane appeared normal. Examination of Dpt–/– stroma by transmission electron microscopy indicated significant disruption of fibril spacing within the posterior lamellae, whereas the mid and anterior regions appeared largely unaffected compared with wild type. The collagen fibrils in Dpt–/– stroma showed a lower fibril volume fraction and a pronounced change in posterior fibrillar organization. There was no apparent difference in fibril diameter between Dpt–/– and wild-type mice. CONCLUSIONS. Collectively, these data suggest that DPT plays a key role in collagen fibril organization. The defects in collagen organization in Dpt–/– cornea appear to be most severe in the posterior stroma. It is possible that DPT interacts with corneal proteoglycans and that this interaction is involved in the maintenance of stromal architecture.