Montelukast-induced increase in IL-25, IL-33, and TSLP expression were suppressed by MAPK inhibitors. A549 cells were pretreated with the MAPK inhibitors SB203580 (SB), SP600125 (SP), and PD98059 (PD) for 1 h and stimulated with montelukast for another 3 h. The supernatant for IL-25, IL-33, and TSLP protein measurement were collected after treatment for 24 h in A549 cells or 96 h in ALI cultures. The montelukast-induced changes in the mRNA (A–C), (n = 5) or protein (D–F), (n = 4) expression of IL-25, IL-33, and TSLP were suppressed by all MAPK inhibitors. The time course of Phospho-p38 (G), phospho-JNK (H), and phospho-ERK (I) levels were determined at 0, 0.5, 1, 3, and 6 h with montelukast treatment in A549 cells (n = 3). The protein levels of IL-25, IL-33, and TSLP by montelukast treatment were decreased by all MAPK inhibitors in ALI cultures (J), (n = 4). Montelukast-induced phospho-p38 (K), phospho-JNK (L), and phospho-ERK (M) levels were increased in ALI cultures (n = 3). The mRNA and protein expression of IL-25, IL-33, and TSLP was detected using real-time PCR or ELISA. MAPKs protein levels were detected using Western blotting. means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 compare with solvent control (Con); # p < 0.05, ## p < 0.01, and ### p < 0.001 compare with montelukast alone (Mon).