Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)D3 and 24R,25(OH)D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR). The purpose of this study was to specifically determine the effects of 1,25(OH)D3 and 24R,25(OH)D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)D3 or 24R,25(OH)D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca (Ca) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)D3 and 24R,25(OH)D3. Cx30 and Cx43 protein levels were also significantly increased in VDR MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)D3 and 1,25(OH)D3. Ca was not affected by 1,25(OH)D3 or 24R,25(OH)D3 in HCEC or MPCEC. We conclude that both 1,25(OH)D3 and 24R,25(OH)D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca.