Gram-negative bacteria use the type 3 secretion system (T3SS) to colonize host cells. T3SSs are ring-shaped macromolecular complexes specific for the transport of effector molecules into host cells. It was recently suggested that a cytosolic ring-shaped protein complex delivers effector molecules to the T3SS. However, how transport of effector proteins is regulated is not known. Here, we report the high-resolution X-ray crystal structure of the whole cytosolic domain of MxiG (MxiG(1-126)), a major component of the inner T3SS rings in Shigella flexneri. MxiG(1-126) folds as an FHA domain, which specifically binds phosphorylated threonines. Indeed, MxiG(1-126) binds to Spa33, a cytoplasmic-ring component of Shigella, as revealed in pulldown studies. Surface plasmon resonance analysis showed specific interaction of MxiG with a Spa33 peptide only if phosphorylated. In total, 24 copies of the MxiG(1-126) crystal structure were fitted into the cryo-EM map of the Shigella T3SS. The phosphoprotein binding site of each MxiG molecule faces the channel of the T3SS, allowing interaction with cytosolic binding partners. Secretion assays and host cell invasion studies of complemented Shigella knockout cells indicated that the phosphoprotein binding of MxiG is essential for bacterial virulence. Our findings suggest that MxiG is involved in T3SS regulation.