We have previously shown that the regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells isolated from fetal lamb under static conditions is positively regulated by PKCdelta. In this study, we explore the role of PKCdelta in regulating shear-induced upregulation of eNOS. We found that shear caused a decrease in PKCdelta activation. Modulation of PKCdelta before shear with a dominant negative mutant of PKCdelta (DN PKCdelta) or bryostatin (a known PKCdelta activator) demonstrated that PKCdelta inhibition potentiates the shear-mediated increases in eNOS expression and activity, while PKCdelta activation inhibited these events. To gain insight into the mechanism by which PKCdelta inhibits shear-induced eNOS expression, we examined activation of STAT3, a known target for PKCdelta phosphorylation. We found that shear decreased the phosphorylation of STAT3. Further the transfection of cells with DN PKCdelta reduced, while PKCdelta activation enhanced, STAT3 phosphorylation in the presence of shear. Transfection of cells with a dominant negative mutant of STAT3 enhanced eNOS promoter activity and nitric oxide production in response to shear. Finally, we found that mutating the STAT3 binding site sequence within the eNOS promoter increased promoter activity in response to shear and that this was no longer inhibited by bryostatin. In conclusion, shear decreases PKCdelta activity and, subsequently, reduces STAT3 binding to the eNOS promoter. This signaling pathway plays a previously unidentified role in the regulation of eNOS expression by shear stress.