The aim of the present study was to explore the etiology of subconjunctival fibrosis (SCF) induced by ethylparaben, the most prevalent preservative in Chinese eye drops. Ethylparaben was administered to the left eyes of male Sprague-Dawley rats in the experimental group twice daily for 1 month, whereas the control group received PBS. Experimental group rats displayed a mild promotion in density of fibroblasts and a tighter deposition of collagen in the bulbar subepithelial connective tissue compared with the control group. Furthermore, the present findings revealed that extracellular matrix expression was promoted in murine bulbar conjunctival tissues in the experimental group. In primary conjunctival fibroblasts, expression of ECM triggered by ethylparaben was suppressed by XAV-939. Furthermore, stimulation of the Wnt/β-catenin axis triggered by ethylparaben was impaired by XAV-939. In conclusion, SCF triggered by ethylparaben results from extra ECM generation of conjunctival fibroblasts via the Wnt/β-catenin axis.