Dry eye syndrome (DES) affects millions of people worldwide. However, as the cellular responses of the corneal epithelium under hyperosmotic stress remain unclear, this study investigated the proteomic changes between human corneal epithelial cells (HCECs) cultured with isosmotic and hyperosmotic media. Under hyperosmotic stress, HCECs increased expressions of sodium-coupled neutral amino acid transporter (SNAT2), glutaminase (GLS-1), and a few isoforms of heat shock protein and aldo-keto reductase family 1. The expressions of SNAT2 and GLS-1 were increased after 6 h of exposure to hyperosmotic stress but not by glutamine deprivation. The hyperosmotic stress increased intracellular levels of glutamine, mitochondrial superoxide, and mitochondrial membrane potential and induced mitochondrial fission in HCECs. Thus, the intracellular level of glutamine was elevated in the hyperosmotic stressed HCECs via the upregulation of SNAT2. Glutamine can act as an osmolyte to regulate the osmolarity of HCECs or be converted to glutamate by GLS-1 for the tricarboxylic acid cycle and oxidative phosphorylation to maintain ATP production under the hyperosmotic stress-induced mitochondrial fission. Thus, the increases in the expressions of SNAT2 and GLS-1 are key osmoregulations in HCECs upon the hyperosmotic stress and may act as corneal biomarkers for monitoring DES progression.