Claudin-2 is highly expressed in human lung adenocarcinoma tissues and cells. Knockdown of claudin-2 decreases cell proliferation and migration. Claudin-2 may be a novel target for lung adenocarcinoma. However, there are no physiologically active substances of foods which decrease claudin-2 expression. We here found that quercetin, a flavonoid present in fruits and vegetables, time- and concentration-dependently decreases claudin-2 expression in lung adenocarcinoma A549 cells. In the present study, we examined what regulatory mechanism is involved in the decrease in claudin-2 expression by quercetin. Claudin-2 expression was decreased by LY-294002, a phosphatidylinositol 3-kinase (PI3-K) inhibitor, and U0126, a MEK inhibitor. These drugs inhibited the phosphorylation of Akt and ERK1/2, which are downstream targets of PI3-K and MEK, respectively. In contrast, quercetin did not inhibit the phosphorylation. Both LY-294002 and U0126 inhibited promoter activity of claudin-2, but quercetin did not. The stability of claudin-2 mRNA was decreased by quercetin. Quercetin increased the expression of microRNA miR-16. An inhibitor of miR-16 rescued quercetin-induced decrease in the claudin-2 expression. These results suggest that quercetin decreases claudin-2 expression mediated by up-regulation of miR-16 expression and instability of claudin-2 mRNA in lung adenocarcinoma cells.