The bacterial type VI secretion system (T6SS), a dynamic organelle, participates in microbial competition by transporting toxic effector molecules to neighbouring cells to kill competitors. TsiV3, a recently defined T6SS immunity protein in Vibrio cholerae, possesses self-protection against killing by T6SS predatory cells by directly binding to and inhibiting their effector protein VgrG-3. Structural information about TsiV3 could help to illuminate its specific mechanism. In this study, TsiV3 from V. cholerae was cloned, expressed and crystallized and single-crystal X-ray diffraction data sets were collected to a resolution of 2.55 Å . Specifically, the crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 73.3, b = 78.12, c = 106.18 Å . Matthews coefficient calculations indicated that the crystal may contain six TsiV3 molecules in one asymmetric unit, with a V M value of 2.25 Å 3 Da À1 and a solvent content of 45.42%.