<jats:title>ABSTRACT</jats:title> <jats:p> Simocyclinone D8 (SD8) exhibits antibiotic activity against gram-positive bacteria but not against gram-negative bacteria. The molecular basis of the cytotoxicity of SD8 is not fully understood, although SD8 has been shown to inhibit the supercoiling activity of <jats:italic>Escherichia coli</jats:italic> gyrase. To understand the mechanism of SD8, we have employed biochemical assays to directly measure the sensitivities of <jats:italic>E</jats:italic> . <jats:italic>coli</jats:italic> and <jats:italic>Staphylococcus aureus</jats:italic> type II topoisomerases to SD8 and microarray analysis to monitor the cellular responses to SD8 treatment. SD8 is a potent inhibitor of either <jats:italic>E</jats:italic> . <jats:italic>coli</jats:italic> or <jats:italic>S</jats:italic> . <jats:italic>aureus</jats:italic> gyrase. In contrast, SD8 exhibits only a moderate inhibitory effect on <jats:italic>S</jats:italic> . <jats:italic>aureus</jats:italic> topoisomerase IV, and <jats:italic>E</jats:italic> . <jats:italic>coli</jats:italic> topoisomerase IV is virtually insensitive to SD8. The antimicrobial effect of SD8 against <jats:italic>E</jats:italic> . <jats:italic>coli</jats:italic> has become evident in the absence of the AcrB multidrug efflux pump. As expected, SD8 treatment exhibits the signature responses to the loss of supercoiling activity in <jats:italic>E</jats:italic> . <jats:italic>coli</jats:italic> : upregulation of gyrase genes and downregulation of the topoisomerase I gene. Unlike quinolone treatment, however, SD8 treatment does not induce the SOS response. These results suggest that DNA gyrase is the target of SD8 in both gram-positive and gram-negative bacteria and that the lack of the antibacterial effect against gram-negative bacteria is due, in part, to the activity of the AcrB efflux pump.