Aortic valve disease initiates on the fibrosa (aorta-facing) side of the valve leaflets, whereas the ventricularis side is disease-protected. Spatially predictable disease correlates with side-specific hemodynamics and gene expression, as fibrosa valve endothelial cells (VECs) experience low, oscillatory shear stress and have lower expression of pathological inhibitors, whereas ventricularis VECs experience high shear stress and have a more protective phenotype. Among the genes expressed in a side-dependent manner, C-type natriuretic peptide (CNP) stands out as a factor that suppresses fibrotic differentiation of aortic valve interstitial cells (VICs). However, the regulation of CNP in aortic valves has not been characterized. In this thesis, shear stress regulation of CNP was studied in vitro using porcine VECs. Steady, high magnitude shear stress and ventricular side-specific shear stress waveforms upregulated NPPC (encoding CNP) in a KLF2-dependent manner. KLF2 and NPPC were upregulated by shear stress in VECs isolated from both the fibrosa and ventricularis sides, but with greater sensitivity in fibrosa-side cells. Notably, while male VECs exhibited side-specific shear stress regulation of KLF2 and NPPC, female VECs did not. Immunohistochemical staining of human valves validated side- and sex-specific CNP expression in normal endothelium. Given the putative role of CNP in valve homeostasis and emerging clinical evidence that stenotic female aortic valves present with more fibrosis than male valves, we hypothesized that the predominance of valvular fibrosis in women would associate with deficiencies in CNP signalling. Here, for the first time, sex differences in CNP expression were demonstrated for human aortic valves:​ female valves, on average, had lower CNP and higher fibrosis upon disease, but the amount of disease (collagen) was not correlated with levels of CNP. In contrast, in diseased regions of male valves, collagen deposition was inversely correlated with CNP, suggesting an anti-fibrotic relationship not present in female valves. Further, treatment of male VICs with CNP suppressed myofibrogenic differentiation in vitro, but female VICs were unresponsive to CNP treatment, indicating deficient CNP signaling. These studies reveal new insights into the hemodynamic regulation of CNP signalling and its role in sex differences of valvular fibrosis.