Background/Objective: A clinical need exists for more effective therapeutics and sustained drug delivery systems to promote ocular surface healing. This study tested the hypothesis that a novel biodegradable, thermoresponsive hydrogel loaded with the human recombinant (rh)MG53 protein, which we have demonstrated to promote corneal healing without fibrosis, would exhibit safety and biocompatibility in vitro and in vivo. Methods: Hydrogel optimization was performed based on varying concentrations of poloxamer 407, poloxamer 188, and hydroxypropyl methylcellulose. Hydrogels were characterized and potential toxicity was evaluated in vitro in cultured corneal epithelium, fibroblasts, and endothelium. In vivo safety and tolerability were assessed in mice and hydrogels were used to evaluate corneal healing following alkali injury. Results: The optimized hydrogel formulation did not result in any detrimental changes to the corneal cells and released functional rhMG53 protein for at least 24 h. In vivo rhMG53-loaded hydrogels improved re-epithelialization, reduced stromal opacification and vascularization, and promoted corneal nerve density. Mechanistically, rhMG53 reduced vascular endothelial cell migration and tube formation by inhibiting pSTAT3 signaling. Conclusions: Taken together, our poloxamer-based thermoresponsive hydrogel effectively released rhMG53 protein and enhanced multiple corneal healing outcomes.