Corneal transparency maintenance relies on the water-pumping function of the corneal endothelium. Currently, corneal transplantation remains the only available treatment for corneal endothelial dysfunction, therefore, the development of alternative therapies is critical due to the global shortage of donor corneas. In our previous study, we confirmed that corneal stromal cells (CSCs) secretion can promote corneal endothelial cells (CEnCs) proliferation. This effect can be enhanced by treatment with lysophosphatidic acid (LPA), a bioactive phospholipid. Nevertheless, the components involved in CSC secretion remain to be elucidated. In this study, we investigated the therapeutic potential of CSC-derived exosomes and exosomal microRNAs (miRNAs) for enhancing CEnCs proliferation and corneal endothelial healing. CSC exosomes were characterized via nanoparticle tracking (NTA), transmission electron microscopy (TEM), and immunoassays. The miRNA expression profiles of CSC exosomes were identified via RNA sequencing, revealing a total of 767 distinct miRNAs. The proliferative effects of CSC exosomes and exosomal miR-221-3p were increased by LPA. Ectopic expression of miR-221-3p further increased CEnC proliferation and suppressed the expression of the CDK inhibitor p27Kip1. The therapeutic efficacy was evaluated using a transcorneal freezing rabbit model, where intrastromal injection of CSC exosomes or ectopic expression of miR-221-3p significantly ameliorated corneal endothelial damage, as supported by improved in vivo corneal recovery, including restoration of corneal thickness, and re-establishment of a hexagonal morphology in the corneal endothelium. Our findings suggest that CSC exosomes and miR-221-3p represent potentially promising cell-free therapies for treating corneal endothelial diseases, highlighting an innovative approach to improving corneal regeneration.