This study investigated the regenerative and healing effect of spirulina on corneal epithelial injury by acid burn in zebrafish. Corneal epithelium of adult zebrafish was evaluated at 0, 12, 24, 48 h after acid burn injury with or without immersion with 0.02 and 0.04 mg/mL of spirulina water extract. The pathological changes of corneal epithelium were assessed by light microscopy. Gene and protein expressions of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP) 9 and MMP13 were determined by immunohistochemistry or real-time PCR. Corneal epithelium was denuded totally after acid burn and gradually regenerated. PCNA-positive cells significantly increased in spirulina treated group. MMP9 and MMP13 mRNA transcripts were significantly decreased in spirulina treated groups. In conclusion, spirulina has regenerative and healing properties by increased keratocyte proliferation, and inhibited extracellular matrix degradation during the regeneration process of corneal epithelial injury by acid burn in zebrafish.