Utilizing the typeⅥ secretion system (T6SS), Pseudomonas aeruginosa secretes the effector protein Tse2, a toxin to other competitive bacteria. It is a newly identified molecular mechanism for P. aeruginosa to win a survival advantage. To avoid being poisoned itself, P. aeruginosa synthesizes the specific immunity protein, Tsi2 to inhibit the toxin. Sequence analysis shows that Tsi2 is a novel antitoxin-like protein which is specific for P. aeruginosa. Using the SAD method, we have successfully resolved the crystal structure of Tsi2 at 1.8Å resolution. Our crystallographic studies reveal that Tsi2 adopts a novel coiled coil conformation which is not found in the antitoxins family previously. Meanwhile, Tsi2 is a well-assembled protein instead of an unfolded protein as an antitoxin in the toxin-free state. Tsi2 functions as a stable dimer and assembles as a unique "clamp" structure through extensive hydrophobic interactions. Two grooves on the dimerization interface combining with one helix of two symmetric molecules respectively imply the potential region interacting with Tse2. This research not only offers comprehensive insights into the molecular essence of Tsi2 as an antitoxin, but also reveals its structural basis of antitoxin activity. Furthermore, the apo Tsi2 structure provides a good framework for further researches on the structure and function of the complex Tse2-Tsi2.