Iron is essential to microorganisms for its important biological function but could be highly toxic in excess. We have used genome-wide transcriptional analysis in Fe3+-treated (4 mM) Bacillus subtilis to reveal the effect of excess Fe3+ on B. subtilis and characterized the potential pathways involved in Fe3+ stress tolerance. A total of 366 and 400 genes were identified as significantly up-regulated and down-regulated, respectively. We found excess Fe3+ had four major influences on B. subtilis: Fe3+ resulted in oxidative stress and induced genes involved in oxidative stress resistance including the SigB-regulated genes, but the PerR regulon was not inducible in Fe3+-mediated oxidative stress except zosA; Fe3+ significantly disturbed homeostasis of Mn2+ and Zn2+, and the mechanism was proposed in this article; the acidity of Fe3+-induced genes involved in acid consuming and production of bases and shifted B. subtilis to carbon starvation state; Fe3+-induced genes related to membrane remodeling (bkd operon), which prevents Fe3+'s incorporation to membrane lipids. Moreover, Fe3+ repressed the stringent control response, consistent with the induction of stringent control in iron limitation, demonstrating that iron might be a signal in stringent control of B. subtilis. This study was the first to provide a comprehensive overview of the genetic response of B. subtilis to ecxess Fe3+.