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Bacterial pathogens often deliver effectors into host cells using type 3 secretion systems (T3SS), the extremity of which forms a translocon that perforates the host plasma membrane. The T3SS encoded by Salmonella pathogenicity island 1 (SPI-1) is genetically associated with an acyl carrier protein, IacP, whose role has remained enigmatic. In this study, using tandem affinity purification, we identify a direct protein-protein interaction between IacP and the translocon protein SipB. We show, by mass spectrometry and radiolabelling, that SipB is acylated, which provides evidence for a modification of the translocon that has not been described before. A unique and conserved cysteine residue of SipB is identified as crucial for this modification. Although acylation of SipB was not essential to virulence, we show that this posttranslational modification promoted SipB insertion into host-cell membranes and pore-forming activity linked to the SPI-1 T3SS. Cooccurrence of acyl carrier and translocon proteins in several γ- and β-proteobacteria suggests that acylation of the translocon is conserved in these other pathogenic bacteria. These results also indicate that acyl carrier proteins, known for their involvement in metabolic pathways, have also evolved as cofactors of new bacterial protein lipidation pathways., Author Summary Acyl carrier proteins are small ubiquitous proteins involved in the synthesis of hydrocarbon based molecules. Notably, they are essential for the synthesis of fatty acids, which are the precursors of membrane phospholipids. They can also be involved in secondary metabolism, for example for the synthesis of molecules with antibacterial properties. Although acyl carrier proteins are widespread, the specific role of each individual protein seems comparatively poorly explored. In this study, we investigate the role of an acyl carrier protein genetically associated with a type 3 secretion system (T3SS). Many Gram-negative bacterial pathogens use T3SS to deliver effectors directly into the cytoplasm of eukaryotic host cells and to subvert host cellular pathways. For this purpose, the translocon, which is the terminal part of T3SS, forms a pore inserted into the host-cell membrane. Here we show that the acyl carrier protein associated with the T3SS has specialized to allow acylation of the translocon. The novel posttranslational modification of the translocon that we describe optimizes insertion into the host-cell membrane and pore-forming activity. This mechanism is likely to be conserved in other pathogenic bacteria given the conserved genetic association between T3SS and acyl carrier protein in several bacteria.