Shigella IcsA is a versatile surface virulence factor required for both early and late pathogenesis stages, extracellularly to intracellularly. Despite IcsA serving as a model Type V secretion system (T5SS) autotransporter to study host pathogen interactions, its detailed molecular architecture is poorly understood. Recently, IcsA was found to switch to a different conformation for its adhesin activity upon sensing of the host stimuli by Shigella Type III secretion system (T3SS). Here, we report that the single cysteine residue (C130) near the N-terminus of IcsA passenger has a role in IcsA adhesin activity. We also show that the IcsA passenger (IcsAp) exists in multiple conformations, and the conformation populations are influenced by a central pair of cysteine residues (C375 and C379). Disruption of either or both central cysteine residues alters the exposure of epitopes to polyclonal anti-IcsA antibodies previously shown to block Shigella adherence, yet without loss of IcsA intracellular functions in actin-based motility (ABM). Anti-IcsA reactivity was restored when the IcsA paired cysteine substitution mutants were expressed in a ∆ipaD background with a constitutively active T3SS, highlighting an interplay between T3SS and T5SS. The work here uncovers a unique molecular switch empowered by centrally localised, short-spaced cysteine pairs in a Type V autotransporter that maintains IcsA’s conformational landscape to aid host immunity evasion.