Purpose To establish a normative database of endothelial cell density (ECD) using in vivo specular microscopy in children under 5 years old. Design Cross-sectional study. Methods Specular microscopy was performed during a clinic visit in cooperative children in the standard upright position. In uncooperative children, specular microscopy was performed in the lateral decubitus position under general anesthesia, before surgery for other reasons. Corneal diameter (CD) was measured in children undergoing general anesthesia and was stratified according to age. Results One hundred and eighteen eyes of 118 patients were included in the study. The mean patient age was 2.6 ± 1.4 years (range 0.1–5 years) and the mean ECD was 3746 ± 370 cells/mm2 (range 3145–5013 cells/mm2). The mean CD under 2 years of age was 11.85 ± 0.57 mm (n = 40, range 10.50–12.75 mm). Up to 2 years of age, ECD was more inversely correlated with CD than with age (r = −0.61, P < .0001; r = −0.38, P = .01, respectively). In contrast, after the age of 2 years, the ECD was inversely correlated with age but not with CD (r = −0.27, P = .02; r = −0.24, P = .2). Between the first and second year of life, the rate of ECD decrease was significantly higher than between 2 and 5 years of age (8.2%, 334 cells/mm2 vs 2.7%, 100 cells/mm2 a year, respectively). Conclusion In the first 2 years of life there is a rapid decline in ECD, which is likely related to growth in CD and hence surface area. After the cornea reaches adult size, the ECD decreases at a rate similar to that reported in adults.