To investigate the effect of calcium ions on promoting the penetrability of riboflavin into the corneal stroma by iontophoresis and to analyse the possible mechanism. Forty rabbits were divided into five groups randomly: 0.1% riboflavin-balanced salt solution (BSS) by iontophoresis group, 0.1% riboflavin-saline solution by iontophoresis group, 0.1% riboflavin-zinc gluconate solution by iontophoresis group, 0.1% riboflavin-calcium gluconate solution by iontophoresis group and classical riboflavin instillation after corneal de-epithelialization as the control group. The riboflavin concentrations in corneal stroma were determined and compared by high-performance liquid chromatography (HPLC) after removing epithelium and endothelium. Iontophoretic delivery of a 0.1% riboflavin-calcium gluconate solution was the closest to the effect of classical de-epithelialization. The other solvents were unsufficient at enhancing the permeability of the riboflavin. Calcium ions can promote the penetrability of riboflavin into the corneal stroma by iontophoresis.