The immune system is very complex, it involves the integrated regulation and expression of hundreds of proteins. To understand in greater detail how the human host defence immunomodulatory peptide LL-37 interacts with innate immunity, a systems approach was pursued. Polychromatic flow cytometry was employed to demonstrate that within human peripheral blood mononuclear cells, CD14+ monocytes, myeloid and plasmocytoid dendritic cells and T- and B-lymphocytes, all responded to LL-37, with the differential production of intracellular cytokines. Microarray analyses with CD14+ monocytes indicated the differential expression of 475 genes in response to stimulation with LL-37. To understand this complex response, bioinformatic interrogation, using InnateDB, of the gene ontology, signalling pathways and transcription factor binding sites was undertaken. Activation of the IkappaBalpha/NFkappaB, mitogen-activated protein kinases p38, ERK1/2 and JNK, and PI3K signalling pathways in response to LL-37 was demonstrated by pathway and ontology over-representation analyses, and confirmed experimentally by inhibitor studies. Computational analysis of the predicted transcription factor binding sites upstream of the genes that were regulated by LL-37 predicted the involvement of several transcription factors including NFkappaB and five novel factors, AP-1, AP-2, SP-1, E2F1, and EGR, which were experimentally confirmed to respond to LL-37 by performing transcription factor array studies on nuclear extracts from LL-37 treated mononuclear cells. These data are discussed as reflecting the integration of several responsive signalling pathways through the involvement of transcription factor complexes in gene expression activated by LL-37 in human mononuclear cells.