Transmembrane protein 16A (TMEM16A), a channel underlying the calcium-activated chloride channel (CaCC) currents, has been shown to be a key regulator of mucus overproduction in airway epithelial cells. However, the precise molecular mechanism involved in the TMEM16A-mediated mucus secretion remains unclear. In the present study, we inquired into a novel signaling mechanism for TMEM16A driving mucin 5AC (MUC5AC) production in human airway epithelial cells. Following treatment for 24-48h with type 13 interleukin (IL-13), an upregulation of TMEM16A expression in both mRNA and protein levels was observed in human bronchial epithelial cell line (HBE16), while signal transducer and activator of transcription 6 (STAT6) inhibition could decrease this elevated expression, suggesting that the regulation of TMEM16A expression by IL-13 was via a STAT6-based transcriptional mechanism. Further investigation of the HBE16 cells revealed that TMEM16A knockdown or specific chloride channel inhibitor T16Ainh-A01 could suppress the CaCC currents and consequently reduce the extracellular regulated kinase (ERK1/2) phosphorylation, accompanying a dramatical decrease in MUC5AC expression. Moreover, pretreated with PD98059, an inhibitor of ERK1/2, the HB16 cells showed a remarkable diminution in TMEM16A-mediated MUC5AC expression. Altogether, STAT6-TMEM16A-ERK1/2 signal pathway and TMEM16A channel activity are required for the IL-13-induced TMEM16A mediated mucus production.