When blood vessels are injured, extracellular matrix is exposed leading to the recruitment and activation of platelets where they aggregate to staunch bleeding. To aggregate, platelets require the release of intracellular calcium linked to the activation of Rap1B. Rap1B is a small GTPase that is activated by guanine nucleotide exchange factors (GEFs) related to Cdc25. CalDAG-GEFI is a Cdc25-related GEF likely to coordinate increased calcium levels with Rap1B activation in platelets. However, the molecular details for how CalDAG-GEFI activates Rap1B are unknown. Here we show that calcium directly regulates CalDAG-GEFI to activate Rap1B. In particular, purified CalDAG-GEFI robustly activated Rap1B using a fluorescence-based nucleotide exchange assay and exchange was lost when CalDAG-GEFI was treated to chelate calcium. Conversely, exchange was fully restored by reintroduction of calcium. Furthermore, substitution within the calcium binding loop of either EF hand crippled the exchange capacity of CalDAG-GEFI. Indeed, substitution in both EF hands abrogated calcium-dependent nucleotide exchange on Rap1B. Many EF hands undergo calcium-dependent conformational rearrangements. To better understand potential conformational rearrangements that regulate CalDAG-GEFI, the differential uptake in deuterium between active CalDAG-GEFI and the form with both EF hands disabled was measured. These studies highlighted calcium-induced conformational rearrangements in two regions: i) both EF hands, and ii) the linker connecting the EF hands and Cdc25 domain. Previous work on CalDAG-GEFII showed that the equivalent linker lies across the surface of the Cdc25 domain needed to engage Ras GTPases and proposed that the linker is displaced from this surface by calcium-dependent rearrangements in the EF hands. To test a similar model for CalDAG-GEFI, the linker was mutated and shown to be required for autoinhibition. In particular, substitution of valine 406 in the linker fully restored exchange capacity of CalDAG-GEFI with both EF hands disabled. Overall, the data support a model whereby the EF hands work in concert, undergoing conformational rearrangements that control the placement of the linker to finely tune the activation of CalDAG-GEFI in platelets. In platelets, CalDAG-GEFI engages Rap1B at the inner leaflet of the plasma membrane. Future studies will define the role of membranes on the calcium-dependent regulation of CalDAG-GEFI.