An antimicrobial protein granulysin is constitutively expressed in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. However, little is known about the precise regulatory mechanisms underlying granulysin gene expression. In this study, we examined the regulatory mechanisms underlying granulysin gene expression using a human monocytic cell line, THP‐1, treated with Acholeplasma laidlawii. The level of granulysin mRNA expression in THP‐1 cells was significantly augmented in response to stimulation with A. laidlawii. The transfection of reporter gene constructs into THP‐1 cells indicated that DNA sequences between residues −329 and −239, relative to the transcriptional start site of the granulysin gene, are responsible for mediating gene induction. In addition, mutagenesis of a putative activator protein‐1 (AP‐1)‐binding site between residues −277 and −271 in the granulysin promoter resulted in the reduction of granulysin promoter activity. Electrophoretic mobility shift assays (EMSA) demonstrated that nuclear extract prepared from A. laidlawii‐treated THP‐1 cells can generate specific binding to DNA oligonucleotides encompassing the AP‐1‐binding site, whereas unstimulated nuclear extract from the cells failed to do so. Furthermore, competition and supershift assays confirmed that A. laidlawii can induce the activation of AP‐1. These results indicate that AP‐1 dominantly participates in the regulation of inducible granulysin gene expression in THP‐1 cells. Therefore, the finding of inducible granulysin gene expression by A. laidlawii suggests that inducible granulysin in macrophages may function as a protective weapon when microbial invasion occurs.