Abstract To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues. Here we show that the Fe-S cluster repair protein, YtfE, contributes to the survival of Y. pseudotuberculosis within the spleen following nitrosative stress. Y. pseudotuberculosis forms clustered centers of replicating bacteria within deep tissues, where peripheral bacteria express the NO-detoxifying gene, hmp . ytfE expression also occurred specifically within peripheral cells at the edges of microcolonies. In the absence of ytfE , the area of microcolonies was significantly smaller than WT, consistent with ytfE contributing to the survival of peripheral cells. The loss of ytfE did not alter the ability of cells to detoxify NO, which occurred within peripheral cells in both WT and Δ ytfE microcolonies. In the absence of NO-detoxifying activity by hmp , NO diffused across Δ ytfE microcolonies, and there was a significant decrease in the area of microcolonies lacking ytfE , indicating that ytfE also contributes to bacterial survival in the absence of NO detoxification. These results indicate a role for Fe-S cluster repair in the survival of Y. pseudotuberculosis within the spleen, and suggest that extracellular bacteria may rely on this pathway for survival within host tissues.