PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates the cell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify the function of PAX6 in human CECs using gene knockout via the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAs for different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but became larger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specific and included keratin 12, keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes were primarily epidermis-related and included keratin 10, keratin 1, involucrin (IVL), filaggrin (FLG). These findings suggest that PAX6 maintains CEC identity by regulating differentiation.