Snake venom Cysteine-Rich Secretory Proteins (svCRiSPs) are important components in the venom of many species of snakes including Viperidae and Elapidae. Although the widespread distribution of svCRiSPs in snake venoms is well known, little is known of the contribution that they make to the local pathophysiology of snakebite. This work aimed to investigate the role of svCRiSPs from the most medically significant species of North American snakes (Crotalus atrox, C. adamanteus, C. scutulatus scutulatus, C. horridus, and Agkistrodon piscivorus), focusing on the cellular and molecular mechanisms underlying vascular biology in snakebite. We evaluated the biological activities of svCRiSPs (Css-CRiSP, Catrox-CRiSP, Cada-CRiSP, Chor-CRiSP, and App-CRiSP) by using both in vitro assays on human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs) permeability and in vivo Miles assay of vascular permeability. Of all the CRiSPs tested, Css-CRiSP and App-CRiSP displayed the highest increase in acute vascular permeability compared to other crotaline CRiSPs. To elucidate the main pathway underlying the endothelial permeability induced by svCRiSPs, we initially screened the changes in protein expression and phosphorylation in HDLECs and HDBECs 30 min after treatment with Css-CRiSP and App-CRiSP using reverse phase protein arrays (RPPA). Stimulating HDBECs with Css-CRiSP and App-CRiSP enhanced caveolin-1 expression. In HDLEC cells, Css-CRiPS and App-CRiSP increased the upregulation of the expression of proteins involved in the phosphoinositide 3-kinase/Akt, Src, MAPK/JNK pathways, cell mobility, and cell adhesion molecules. These preliminary observations suggest that Css-CRiSP and App-CRiSP induce the increased endothelial monolayer permeability in HDBECs and HDLECs via different mechanisms. Knowledge gained from these studies provides insights into the molecular mechanisms that underlie the effects of svCRiSPs on vascular function and contributes to a new level of understanding of the pathophysiology of snakebite.