Mechanical stimulation plays an important role in the process of keratoconus after LASIK. However, the mechanism is still unclear. In this study, the effects of mechanical stretch on the expression of aquaporin 1(AQP1) and its downstream pathways were explored in human keratocytes, which would provide a reference for further elucidating the mechanism of keratoconus. Human keratocytes were subjected to Ca2+ chelator BAPTA treatment, or cyclic mechanical stretch with FLEX-4000. The intracellular Ca2+ concentration and cAMP content were detected by Fluo-3 fluorescent probe and ELISA respectively. The expression of AQP1 and the related genes in the downstream signaling pathway was analyzed by real-time PCR. The results show that the expression of AQP1 and its downstream genes in FAK and Wnt pathways was induced by mechanical stretch in human keratocytes. Besides, the intracellular Ca2+ and cAMP contents increased significantly after mechanical stretch. BAPTA could inhibit the stretch-induced expressions of AQP1 and its downstream genes including ARHGEF2, Wnt11, and MMP2. The results indicate that mechanical stretch could induce the expression of AQP1 in human keratocytes by regulating intracellular Ca2+ and cAMP contents, and further participate in the regulation of MMP2-related extracellular matrix metabolism through FAK and Wnt pathways.