Sodium azide inhibits bacterial growth by inhibiting the activity of SecA, an ATPase required for translocation of proteins across the cytoplasmic membrane. To investigate the mechanism of action of azide, we used transposon directed insertion-site sequencing (TraDIS) to screen a high-density library of transposon insertion mutants for mutations that affect the susceptibility of E. coli to azide. Insertions in genes encoding most components of the Sec machinery increased susceptibility to azide. However, insertions truncating the C-terminal extension (CTE) of SecA decreased susceptibility of E. coli to azide. Insertions in genes encoding many metal binding proteins also increased susceptibility to azide, and transcriptional profiling suggested that treatment with azide disrupted iron homeostasis. The presence of iron in the media decreased the susceptibility of E. coli to azide, and mutations in the secA gene that confer resistance to azide altered the response of E. coli to iron limitation, suggesting a connection between iron metabolism and protein translocation. Although previous work suggests that SecA binds to zinc, SecA copurified with iron when expressed at physiological levels, and azide disrupted the interaction of the C-terminal metal-binding domain (MeBD) with iron in vivo. Biophysical analysis of metal binding by the MeBD using isothermal titration calorimetry and 1H-nuclear magnetic resonance indicated a clear binding preference for Fe2+ over Zn2+. These results indicate that the physiological ligand of SecA is iron and that azide inhibits SecA by disrupting iron binding. Importance: Sodium azide is a common preservative that inhibits bacterial growth by inhibiting SecA, an ATPase that is required for transporting proteins across the cytoplasmic membrane. Previous studies have suggested that azide inhibits the ATPase activity SecA by slowing the rate of nucleotide exchange. However, our results suggest that azide inhibits SecA by disrupting the structure of the C-terminal MeBD. It is thought that this domain binds to Zn2+. However, our results indicate that the MeBD normally binds to Fe2+ and that azide disrupts the interaction of the MeBD with Fe2+. Furthermore, mutations in the secA gene that confer azide resistance also cause a defect in the response of E. coli to iron depletion. SecA-dependent protein translocation is generally thought to be unregulated. However, these results suggest that the activity of SecA could be regulated in response to iron limitation.