It has been reported that overexpression of MUC5AC induced by excessive inflammation leads to airway obstruction in respiratory diseases such as chronic obstructive pulmonary disease and asthma. 15-Hydroxyeicosatetraenoic acid (15-HETE) has been reported to have anti-inflammatory effects, but the role of 15-HETE in respiratory inflammation has not been determined. Therefore, the aim of this study was to investigate the effects of 15-HETE on MUC5AC expression and related pathways. In this study, phorbol-12-myristate-13-acetate (PMA) was used to stimulate NCI-H292 bronchial epithelial cells in order to examine the effects of 15-HETE. 15-HETE inhibited PMA-induced expression of MUC5AC mRNA and secretion of MUC5AC protein. Also, 15-HETE regulated matrix metallopeptidase 9 (MMP-9), mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK). In addition, 15-HETE decreased translocation of specificity protein-1 (Sp-1) transcription factor and nuclear factor kappaB (NF-κB) into nuclear. Furthermore, 15-HETE enhanced transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) as a PPARγ agonist. This activity reduced phosphorylation of protein kinase B (PΚB/Akt) by increasing the expression of phosphatase and tensin homolog (PTEN). In conclusion, 15-HETE regulated MUC5AC expression via modulating MMP-9, MEK/ERK/Sp-1 and PPARγ/PTEN/ Akt signaling pathways in PMA-treated respiratory epithelial cells.