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PURPOSE The aim of this study was to characterize the regulatory role of microRNA-29a (miR-29a) in myopia, providing support for potential biomarkers and new therapeutic targets of myopia in humans. METHODS The miR-29a expression level was detected in the aqueous humor and peripheral blood plasma of 21 high myopic patients and eight cataract control patients using quantitative polymerase chain reaction. iTRAQ analysis of proteomes was conducted to show the regulatory effect of miR-29a on human scleral fibroblasts (SFs) cultured in vitro. We also assessed proliferation, migration, and collagen I synthesis in SF cells, mediated by miR-29a. RESULTS MiR-29a expression was significantly higher in the aqueous humor of highly myopic patients than in the cataract control patients (fold change: 4.861, p = 0.001). miR-29a inhibited the synthesis of type I collagen in human SF cells and enhanced cell migration, but had no significant effect on cell proliferation. CONCLUSION MiR-29a was highly expressed in aqueous humor of myopia patients and inhibited the synthesis of type I collagen in human SF cells in vivo, thereby it may play an important role in myopia development.