Astragalus polysaccharides (APS), one of the major active components in Astragalus membranaceus, is an effective immunomodulator used in the treatment of immunological diseases in China. However, the anti-infective action and mechanism of APS is not fully known. In the present study, we found that APS induced the expression of human cathelicidin antimicrobial peptide LL-37, a key host anti-infective molecule, in both mRNA and protein levels in respiratory epithelial cells HBE16 and A549. Furthermore, the lysate and supernatant from APS-treated HBE16 cells both exhibited an obvious antibacterial action, which was partially neutralizated by LL-37 monoclonal antibody. In addition, APS also significantly elevated the phosphorylation of p38 MAPK and JNK and caused the degradation of IκBα. Specific inhibitors of p38 MAPK, JNK, or NF-κB obviously abolished APS-induced LL-37 synthesis and antibacterial activity, respectively. Taken together, our results confirmed the enhancement of APS on LL-37 induction and antibacterial action in respiratory epithelial cells, which may be attributed to activation of p38 MAPK/JNK and NF-κB pathways. Furthermore, these results also supported the clinical application of APS in the treatment of infectious diseases.