Problem statement: Flow mediated dilation is compromised in cystic fibrosis (CF), demonstrating endothelial dysfunction in response to shear stress. Inhibition of endothelial CF-transmembrane conductance regulator (CFTR) activity causes a pro-inflammatory and pro-oxidant state. Inflammation and oxidative stress cause endothelial barrier dysfunction by re-organising the endothelial actin cytoskeleton. It is currently unknown, however, whether CFTR inhibition causes F-actin polymerisation. The present study, therefore, aimed to investigate the implications of CFTR inhibition on F-actin polymerisation and morphological alignment following exposure to shear stress in human lung microvascular endothelial cells (HLMVEC) in vitro. Methods: HLMVEC were cultured in the absence and presence of the pharmacological CFTR inhibitor GlyH-101 (20 µM), during 16 h, 24 h and 48 h of exposure to shear stress (11.1 dynes/cm2) or static conditions. Nitrite and endothelin (ET)-1 were analysed in cell media supernatants. Morphological changes in response to shear stress were detected using Crystal Violet staining. Cortical rim, perinuclear and stress fibre F-actin reformation were detected using Rhodamine Phalloidin (red) and Hoechst 33342 (blue) staining. Values are means ± SEM. Results: Exposure to 48 h of shear stress increased [nitrite] (+146.2 ± 2.0% vs. static conditions; p < 0.05), whereas 48 h (-57.9 ± 3.6% vs. static conditions; p < 0.05) and 24 h (-63.5 ± 5.6% vs. static conditions; p < 0.05) of shear stress lowered [ET-1] in HLMVEC supernatant. Notably, 16 h or 24 h of shear stress had no significant effect on [nitrite], nor did 16 h of shear stress significantly affect [ET-1] (p > 0.05). The addition of GlyH-101 did not alter [nitrite] or [ET-1] (all p > 0.05). Shear stress over 48 h induced cellular alignment with the direction of flow; however, the addition of GlyH-101 prevented such alignment (Figure 1). GlyH-101 increased cytosolic F-actin polymerisation under static conditions (p < 0.05). GlyH-101 also caused a re-distribution of F-actin from the cortical rim to cytosolic stress fibres under both static and shear stress conditions over 24 h (Figure 2). Conclusions: Functional CFTR appears to limit cytosolic F-actin polymerisation, while maintaining a cortical rim distribution that is important for cellular integrity and CFTR function. Inhibition of CFTR, with increased F-actin stress fibre formation, prevented HLMVEC alignment with flow independent of [nitrite] and [ET-1]. CFTR, therefore, plays an important role in the regulation of actin dynamics in HLMVEC and may contribute to the endothelial perturbations that are evident even in relatively mild CF.