Gram-negative bacterial proteins secreted by ABC exporters carry a secretion signal in their carboxylic extremities. This characteristic suggests that the polypeptide needs to be fully synthesized before it can be secreted and, therefore, presumably may fold at least in part before its secretion. We investigated the relationship between folding and secretion using HasA, a hemoprotein of Serratia marcescens secreted into the extracellular medium by a dedicated Has ABC exporter. We first demonstrated that when HasA is sequestered in the cytoplasm it can acquire its tertiary structure, as assessed from its capacity to bind heme. The cytoplasmic pool of HasA cannot be secreted and inhibits the secretion of newly synthesized molecules. HasA folding in the cytoplasm was independent of either its capacity to bind heme or the presence of SecB, although SecB is essential for HasA secretion. Our findings indicate a strong coupling between synthesis and secretion in the type I secretion pathway.