Connexin37 (Cx37) is a gap junction protein essential for cell-cell communication in the vascular system. We have recently shown that Cx37, which is expressed in endothelial cells, platelets and megakaryocytes, limits thrombus propensity by permitting cAMP signaling between aggregating platelets. Here, we have performed high throughput phage display to identify potential binding partners for the intracellular regulatory C-terminus of Cx37 (Cx37CT). We retrieved 2 consensus binding motifs for Cx37CT: WHK_[K,R]XP_ and FHK_[K,R]XXP_ Sequence alignments against the NCBI protein database indicated 80% homology of one of the selected peptides with FVIII B-domain. We performed cross-linking reactions using BS3 and confirmed that an 11-mer peptide of the FVIII B-domain sequence linked to recombinant Cx37CT. In vitro binding of this peptide to Cx37CT was also confirmed by surface plasmon resonance. The dissociation constant of Cx37CT to FVIII B-domain peptides was ∼2 × 10-5 M. Others peptides sequences, designed upstream or downstream the FVIII B-domain sequence, showed a very low or no affinity for Cx37CT. Finally, in vivo studies revealed that thrombin generation in platelet-poor plasma of Cx37-/- mice (endogenous thrombin potential, 634 ± 11 nM min, mean ± SEM) was increased compared to Cx37+/+ mice (427 ± 12, P < 0.001). Moreover, partial activated thromboplastin time (aPTT) was shorter in Cx37-/- (39.7 ± 1.5 s) than in Cx37+/+ mice (45.9 ± 1.8, P = 0.03), whereas prothrombin time was comparable. The shorter aPTT in Cx37-/- mice correlated with higher circulating FVIII activity (46.0 ± 0.7 vs. 53.5 ± 2.7 s for Cx37+/+, P = 0.03). Overall, our data show for the first time a functional interaction between FVIII and Cx37. This interaction may be relevant for the control of FVIII secretion and, thereby, in the regulation of levels of FVIII circulating in blood. In addition, results of this study may open new perspectives to improve the efficiency of recombinant FVIII manufacturing.