Bordetella pertussis is the causative agent of pertussis (whooping cough), a contagious respiratory disease that has recently seen a resurgence despite high vaccination coverage, necessitating improvement of current pertussis vaccines. An autotransporter of B. pertussis, virulence-associated gene 8 (Vag8), has been proposed as an additional component to improve pertussis vaccines. Vag8 is known to play a role in evasion of the complement system and activation of the contact system by inactivating the complement regulating factor, C1 inhibitor (C1 Inh), which inhibits serine proteases, such as plasma kallikrein (PK). However, the nature of the molecular interaction between Vag8 and C1 Inh remains to be determined. In the present study, we attempted to determine the minimum region of Vag8 that interacts with C1 Inh by examining the differently–truncated Vag8 derivatives for the ability to bind and inactivate C1 Inh. The region of Vag8 from amino–acid residues 102 to 548 was found to bind C1 Inh and cancel its inhibitory action on the protease activity of PK at the same level as a Vag8 fragment from amino–acid residues 52 to 648 covering the passenger domain, which carries its extracellular function. In contrast, the truncated Vag8 containing amino–acid residues 102 – 479 or 202 – 648 barely interacted with C1 Inh. These results indicated that the two separate regions of amino–acid residues 102 – 202 and 479 – 548 are likely required for the interaction with C1 Inh. Importance Pertussis is currently reemerging worldwide, and is still one of the greatest disease burdens in infants. B. pertussis produces a number of virulence factors, including toxins, adhesins, and autotransporters. One of the autotransporters, Vag8, which binds and inactivates the complement regulator C1 Inh, is considered to contribute to the establishment of B. pertussis infection. However, the nature of the interaction between Vag8 and C1 Inh remains to be explored. In this study, we narrowed down the region of Vag8 that interacts with C1 Inh and demonstrated that at least two separate regions of Vag8 are necessary for the interaction with C1 Inh. Our results provide insight into the structure–function relationship of the Vag8 molecule and information to determine its potential role in the pathogenesis of B. pertussis.