Osteomyelitis often causes functional impairment due to tissue destruction. This report demonstrates a novel previously unappreciated role of osteoblasts. Samples of osteomyelitic bone and bacterially challenged osteoblasts produce increased amounts of antimicrobial peptides in order to combat bacterial bone infection. An osteomyelitis mouse model confirmed the osseous induction of the murine homologue of human β‐defensin‐2, suggesting a central role in the prevention of bacterial bone infection. Antimicrobial peptides are effectors of the innate defence system and play a key role in host protection at cellular surfaces. Some of them are produced constitutively, whereas others are induced during infection. Human β‐defensins represent a major subclass of antimicrobial peptides and act as a first line of defence through their broad spectrum of potent antimicrobial activity. The aim of the present in‐vitro and in‐vivo investigations was to study the expression and regulation of human β‐defensin‐2 in the case of bacterial bone infection and to analyse the effects of immunosuppressive drugs on bone‐derived antimicrobial peptide expression. Samples of healthy human bone, osteomyelitic bone and cultured osteoblasts (hFOB cells) were assessed for the expression of human β‐defensin‐2. Regulation of human β‐defensin‐2 was studied in hFOB cells after exposure to bacterial supernatants, proinflammatory cytokines and immunosuppressive drugs (glucocorticoids and methotrexate) and was assayed by enzyme‐linked immunosorbent assay. An osteomyelitis mouse model was performed to demonstrate the regulation of the murine homologue of human β‐defensin‐2, named murine β‐defensin‐3, by real‐time reverse transcription‐polymerase chain reaction and immunohistochemistry. Healthy human bone and cultured osteoblasts are able to produce human β‐defensin‐2 under standard conditions. Samples of infected bone produce higher levels of endogenous antibiotics, such as human β‐defensin‐2, when compared with samples of healthy bone. A clear induction of human β‐defensin‐2 was observed after exposure of cultured osteoblasts to Gram‐positive bacteria or proinflammatory cytokines. Additional treatment with glucocorticoids or methotrexate prevented bacteria‐mediated antimicrobial peptide induction in cultured osteoblasts. The osteomyelitis mouse model demonstrated transcriptional upregulation of the murine homologue of human β‐defensin‐2, namely murine β‐defensin‐3, in bone after intraosseous contamination of the tibia. Human and murine bone have the ability to produce broad‐spectrum endogenous antibiotics when challenged by micro‐organisms in vitro and in vivo. Immunosuppressive drugs, such as glucocorticoids or methotrexate, may increase the susceptibility to bone infection by decreasing antimicrobial peptide expression levels in case of microbial challenge. The induction of human β‐defensin‐2 following bacterial contact suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection.