Tight junctions (TJs) are cell-adhesion structures responsible for the epithelial barrier. We reported that accumulation of cholesterol at the apical junctions is required for TJ formation [K. Shigetomi, Y. Ono, T. Inai, J. Ikenouchi, , 2373-2381 (2018)]. However, it is unclear how cholesterol accumulates and informs TJ formation-and whether cholesterol enrichment precedes or follows the assembly of claudins in the first place. Here, we established an epithelial cell line (claudin-null cells) that lacks TJs by knocking out claudins. Despite the lack of TJs, cholesterol normally accumulated in the vicinity of the apical junctions. Assembly of claudins at TJs is thought to require binding to zonula occludens (ZO) proteins; however, a claudin mutant that cannot bind to ZO proteins still formed TJ strands. ZO proteins were however necessary for cholesterol accumulation at the apical junctions through their effect on the junctional actomyosin cytoskeleton. We propose that ZO proteins not only function as scaffolds for claudins but also promote TJ formation of cholesterol-rich membrane domains at apical junctions.