Aim: This study aimed to investigate acute corneal toxicity of commercially available diquafosol 3% ophthalmic solution (Diquas®), which contains C12 benzalkonium chloride (BAC) as a preservative. Methods: Corneal transepithelial electrical resistance (TER) changes after a 60-second exposure to Diquas® (diquafosol 3% preserved with 0.0075% C12 BAC); 0.0075% C12 BAC and 0.0075% C12, C14, C16 BAC mixture were measured in living rabbits. Corneal damage was also examined by scanning electron microscopy (SEM). Hank's balanced salt solution (HBSS) was used as a control. Results: Diquas® and 0.0075% C12 BAC did not produce any significant decrease in the corneal TER as compared to the HBSS control eyes. There was a significant decrease in the corneal TER after exposure of the cornea to the 0.0075% C12, C14, C16 BAC mixture (p < 0.01). SEM revealed that the superficial cells of the corneas exposed to the 0.0075% BAC mixture were damaged and exhibited degenerated microvilli. Conversely, the superficial cells of corneas exposed to Diquas® or 0.0075% C12 BAC appeared normal and had normal microvilli under SEM examinations. Conclusion: The acute corneal toxicity of Diquas® is less than that of the 0.0075% BAC mixture. Diquas® preserved with 0.0075% C12 BAC did not show acute corneal toxicity.