AIM: To quantify corneal ultrastructure using laser scanning in vivo confocal microscopy (IVCM) in patients with keratoconus and control subjects. • METHODS: Unscarred corneas of 78 keratoconic subjects without a history of contact lens use and 36 age-matched control subjects were evaluated with slit-lamp examination (SLE), corneal topography and laser scanning IVCM. One eye was randomly chosen for analysis. Anterior and posterior stromal keratocyte, endothelial cell and basal epithelial cell densities and sub-basal nerve structure were evaluated. • RESULTS: IVCM qualitatively demonstrated enlarged basal epithelial cells, structural changes in sub-basal and stromal nerve fibers, abnormal stromal keratocytes and keratocyte nuclei, and pleomorphism and enlargement of endothelial cells. Compared with control subjects, significant reductions in basal epithelial cell density (5817±306 cells/mm2 vs 4802±508 cells/mm2, P<0.001), anterior stromal keratocyte density (800±111 cells/mm2 vs 555±115 cells/mm2, P<0.001), posterior stromal keratocyte density (333±34 cells/mm2 vs 270±47 cells/mm2, P<0.001), endothelial cell density (2875±223 cells/mm2 vs 2686±265 cells/mm2, P< 0.001), sub-basal nerve fiber density (31.2±8.4 nerves/mm2 vs 18.1±9.2 nerves/mm2, P<0.001), sub-basal nerve fiber length (21.4±3.4 mm/mm2 vs 16.1±5.1 mm/mm2,P<0.001), and sub-basal nerve branch density (median 50.0 (first quartile 31.2 - third quartile 68.7) nerve branches/mm2 vs median 25.0 (first quartile 6.2 - third quartile 45.3) nerve branches/mm2, P<0.001) were observed in patients with keratoconus. • CONCLUSION: Significant microstructural abnormalities were identified in all corneal layers in the eyes of subjects with keratoconus using IVCM. This non-invasive in vivo technique provides an important means to define and follow progress of microstructural changes in patients with keratoconus.