<b><i>Background:</i></b> Mucus overproduction is one of the major pathological features of asthma, and MUC5AC is the major mucin component of airway mucus. However, whether Notch signaling is implicated in the regulation of MUC5AC expression in airway secretary cells is still undetermined. <b><i>Objective:</i></b> The aim of this study is to examine whether Notch signaling can regulate MUC5AC expression and explore the molecular mechanisms. <b><i>Methods:</i></b> Mouse mtCC1-2 cells and human NCI-H292 cells were transfected with NIC, and MUC5AC expression was examined. Using gene reporter assays, site-directed mutagenesis, and ChIP assays, the activity of both mouse and human <i>MUC5AC</i> promoter was analyzed. <b><i>Results:</i></b> Notch signaling regulated MUC5AC expression both in mouse mtCC1-2 cells and in human NCI-H292 cells. Several Hes-binding site N-boxes were identified in the 5′ region of both mouse and human <i>MUC5AC</i> promoters. Overexpression of NIC resulted in activation of the <i>MUC5AC</i> promoter. Site-directed mutagenesis report assays revealed that Hes proteins might repress both mouse and human <i>MUC5AC</i> promoter activity. Furthermore, ChIP assays confirmed that Hes1 binds to the <i>MUC5AC</i> promoter both in mouse mtCC1-2 cells and in human NCI-H292 cells. <b><i>Conclusions:</i></b> Notch signaling can directly downregulate <i>MUC5AC</i> promoter activity through Hes1-dependent mechanisms, which may be identified as possible targets for pharmacotherapy of airway mucus hypersecretion in asthma.