In response to iron limitation, the siderophore enterobactin is synthesized and secreted by Escherichia coli. Its biosynthesis is performed by a series of enzymes encoded by the Ent gene cluster. Among the genes of this cluster, ybdB has not been implicated in enterobactin production to date. We demonstrate here an in vivo role for the hotdog protein EntH (YbdB) in the optimal production of enterobactin. Indeed, we showed that EntH is a thioesterase specifically produced under iron limitation conditions. Furthermore, EntH interacts specifically with the aryl carrier protein (ArCP) domain of EntB, a crucial bifunctional enzyme of the enterobactin biosynthesis pathway and a potential target of EntH thioesterase activity. A strain devoid of EntH is impaired for growth under iron limitation associated with the presence of the salicylate inhibitor, correlating with the diminution of enterobactin production under these conditions. Normal growth and enterobactin production are restored upon expression of entH in trans. Inversely, unnecessary overproduction of EntH provokes a fall of the quantity of siderophore produced under iron starvation conditions. Our findings point to a proofreading role for EntH during biosynthesis of enterobactin in vivo. EntH thioesterase activity could be required for cleaving wrongly charged molecules on the carrier protein EntB. This is the first description of such a role in the optimization of a nonribosomal biosynthesis pathway for a protein of the hotdog superfamily.