Purpose: To evaluate the corneal microstructure in keratoconus with in vivo confocal microscopy (IVCM). Methods: Unscarred corneas of 68 patients with keratoconus and 22 controls were evaluated with slit lamp examination, corneal topography, and IVCM (Confoscan 3.0, Vigonza, Italy). One eye was randomly chosen for analysis. Keratocyte, endothelial cell and basal epithelial densities, sub-basal and stromal nerve structure, and severity of stromal haze were evaluated. Results: Compared with corneas of control subjects, patients with keratoconus had a significantly lower anterior stromal keratocyte density (1279 197 cells/mm2 vs. 1132 178 cells/mm2, p = 0.002), lower midstromal keratocyte density (904 213 cells/mm2 vs. 770 120 cells/mm2, p 0.001), lower posterior stromal keratocyte density (935 113 cells/mm2 vs. 725 113 cells/mm2, p 0.001), lower endothelial cell density (2924 300 cells/mm2 vs. 2719 279 cells/mm2, p = 0.004), lower basal epithelial cell density (5987 699 cells/mm2 vs. 4365 537 cells/mm2, p 0.001), lower sub-basal long nerve density (32.0 6.5 nerves/mm2 vs. 19.6 6.5 nerves/mm2, p 0.001), thicker sub-basal (3.2 0.4 m vs. 3.7 1.1 m, p = 0.01) and stromal nerves (5.0 1.2 m vs. 8.0 2.9 m, p 0.001), and higher proportion of corneas with haze (40.9% vs. 92.6%, p 0.001). A history of contact lens use (n = 12) was not associated with lower keratocyte, endothelial cell, or basal epithelial cell counts. Conclusions: Corneal microstructure is abnormal in patients with keratoconus. Keratocyte and endothelial cell loss appears to be present in keratoconic corneas.