During bacterial infections, antimicrobial peptides are synthesised as an important part of the innate immune system. However, expression and function in the central nervous system (CNS) need further investigations. The aim of this study was to examine the involvement of the pattern-recognition-receptor toll-like receptor 9 (TLR9) in the expression of the cathelin-related antimicrobial peptide (CRAMP) and to characterise the participating signal transduction pathways. In primary TLR9 deficient and wildtype mice astrocytes as well as microglia cells, the expression of CRAMP after treatment with the TLR9 agonist unmethylated cytosine-guanine oligodeoxynucleotide motifs (CpG-DNA) was examined in vitro. In vivo CRAMP expression after intraventricular infusion of CpG-DNA in TLR9 deficient and wildtype mice as well as in mice with pneumococcal meningitis localised in glial cells was determined. Furthermore, the regulation of different signal transduction pathways involved in CpG-DNA-induced CRAMP expression in glial cells was analysed. An in vitro and in vivo CpG-DNA-induced increase of CRAMP expression in astrocytes and microglia cells using real time RT-PCR and immunofluorescence was demonstrated. Different signal transduction pathways such as mitogen-activated protein kinases and inflammatory mediated pathways are involved in the expression of CRAMP in primary glial cells. Interestingly, TLR9-deficient glial cells showed a reduced but not completely abolished CRAMP mRNA expression and ERK1/2 phosphorylation in response to CpG-DNA treatment. On the other side in vivo, TLR9 deletion did not change CRAMP expression after bacterial infection. In conclusion, our results show that TLR9 can induce the expression of antimicrobial peptides such as CRAMP in response to bacterial DNA motifs in primary glial cells. Additional findings suggest also that CpG-DNA-induced effects are not only mediated by TLR9, but also mediated by other pattern recognition receptors.