Spermine (Spm), a potent bactericidal polyamine, exerts a strong synergistic effect with beta-lactams against methicillin-resistant Staphylococcus aureus (MRSA). To explore the Spm-based antibacterial targets in S. aureus, time course-dependent transcriptome analysis was conducted on Mu50 (MRSA) in the absence and presence of Spm. Genes in the sigB regulon and most ATP-producing pathways were found down-regulated when exposure to high dose Spm. In contrast, a number of genes for iron acquisition and regulation showed significant induction, indicating a specific connection between Spm and iron-depletion. The tetM gene for tetracycline (Tc) resistance exhibited most significant fold change among the listed genes. It was specifically upregulated by Tc and Spm but not by other ribosome-targeted drugs or other polyamines; however, such induction of tetM cannot confer resistance to Spm. A set of genes for osmotic balance, including kdpABCDE for potassium ion uptake and regulation, was also induced by Spm stress. Addition of KCl or NaCl, but not high concentration sucrose, was found to increase Spm MIC over 30-fold. In summary, transcriptome analysis demonstrated a specific pattern of response upon Spm exposure, suggesting Spm may alter the intracellular iron status and suppress the SigB regulon to exert its toxicity.