Background: Factor V is an inactive procofactor. Removal of its B-domain drives activation through an undefined mechanism. Results: Basic and acidic regions within the B-domain impair factor Xa binding and cofactor function. Conclusion: Autoinhibitory B-domain segments maintain the procofactor state and their disruption activates FV. Significance: The study provides insights into how FV activation unfolds and defines an unexpected sequence-specific function of the B-domain. Activation of blood coagulation factor V (FV) is a key reaction of hemostasis. FV circulates in plasma as an inactive procofactor, and proteolytic removal of a large central B-domain converts it to an active cofactor (FVa) for factor Xa (FXa). Here we show that two short evolutionary conserved segments of the B-domain, together termed the procofactor regulatory region, serve an essential autoinhibitory function. This newly identified motif consists of a basic (963–1008) and an acidic (1493–1537) region and defines the minimal sequence requirements to maintain FV as a procofactor. Our data suggest that dismantling this autoinhibitory region via deletion or proteolysis is the driving force to unveil a high affinity binding site(s) for FXa. These findings document an unexpected sequence-specific role for the B-domain by negatively regulating FV function and preventing activity of the procofactor. These new mechanistic insights point to new ways in which the FV procofactor to cofactor transition could be modulated to alter hemostasis.