PURPOSE: The emerging epitranscriptomics offers insights into the physiopathological roles of various RNA modifications. The RNA methylase NOP2/Sun domain family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modification of mRNAs. However, the role of NSUN2 in corneal epithelial wound healing (CEWH) remains unknown. Here we describe the functional mechanisms of NSUN2 in mediating CEWH. METHODS: RT-qPCR, Western blot, dot blot, and ELISA were used to determine the NSUN2 expression and overall RNA m5C level during CEWH. NSUN2 silencing or overexpression was performed to explore its involvement in CEWH both in vivo and in vitro. Multi-omics was integrated to reveal the downstream target of NSUN2. MeRIP-qPCR, RIP-qPCR, and luciferase assay, as well as in vivo and in vitro functional assays, clarified the molecular mechanism of NSUN2 in CEWH. RESULTS: The NSUN2 expression and RNA m5C level increased significantly during CEWH. NSUN2 knockdown significantly delayed CEWH in vivo and inhibited human corneal epithelial cells (HCEC) proliferation and migration in vitro, whereas NSUN2 overexpression prominently enhanced HCEC proliferation and migration. Mechanistically, we found that NSUN2 increased ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) translation through the binding of RNA m5C reader Aly/REF export factor. Accordingly, UHRF1 knockdown significantly delayed CEWH in vivo and inhibited HCEC proliferation and migration in vitro. Furthermore, UHRF1 overexpression effectively rescued the inhibitory effect of NSUN2 silencing on HCEC proliferation and migration. CONCLUSIONS: NSUN2-mediated m5C modification of UHRF1 mRNA modulates CEWH. This finding highlights the critical importance of this novel epitranscriptomic mechanism in control of CEWH.