Mucin overproduction is a hallmark of chronic respiratory diseases (CRD) such as chronic obstructive pulmonary disease and asthma, and otitis media. Despite the fact that nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae are co-existing under these disease conditions, little is known about how NTHi and S. pneumoniae induce mucin overproduction. Here we show that NTHi and S. pneumoniae, when present together, synergistically induce MUC5AC mucin transcription. TLR2/4-MyD88-TAK1 signaling cascade transmits signal to regulate the synergistic induction of MUC5AC. The activation of MKK3/6-p38 and ERK MAPK pathways are required for the synergistic induction of MUC5AC. Moreover, S. pneumoniae synergizes with NTHi to induce MUC5AC expression via AP-1-dependent mechanism. Thus, our studies provide direct evidence for the synergistic induction of MUC5AC in mixed infections and bring novel insights into our understanding of molecular mechanisms underlying polymicrobial infections in CRD and OM.