The endothelium is an interconnected network of cells whose configuration is critical to the organ’s ability to sense mechanical forces. Haemodynamic forces act on blood vessels via changes in endothelial [Ca2+]i to evoke a diversity of biological responses, which includes the control of arterial tone, the extent of smooth muscle proliferation and vascular remodelling. However studying endothelial [Ca2+]i in intact and pressurised blood vessels is challenging (particularly in the larger arteries in which atherosclerosis and re-stenosis occurs) because of the thickness of the medial and adventitial layers. To overcome the difficulty, we developed an imaging system, using gradient index (GRIN) lens technology, to visualise the endothelium from inside arteries at normal physiological pressures where the arteries’ structural integrity is maintained. Our results show that mechanotransduction may occur via changes in InsP3-evoked Ca2+ signals. Segments of common carotid arteries from male Wistar rats (150-250 g) were removed and mounted in a custom-built ateriograph superfused with MOPS buffered saline (37°C). The Ca2+-sensitive fluorescent indicator Oregon Green BAPTA-1 AM (20 µM) was loaded preferentially into endothelial cells and visualised with a custom built, side-viewing imaging probe based on a GRIN relay lens. The imaging probe was optically coupled to a custom-built fluorescence microscope, permitting direct visualisation of an area encompassing ~200 endothelial cells. The artery was pressurised (60-160 mmHg) and endothelial [Ca2+]i measured on a per-cell basis, using a custom image processing procedure. To allow the total response from all responding cells to be accurately examined, a custom analysis script (Python programming language) was used to differentiate, and then align (in time) the response of individual cells. ACh evoked increases in [Ca2+]i that were sensitive to 2-aminoethoxydiphenyl borate (100 µM) and cyclopiazonic acid (100 µM), suggesting the [Ca2+]i rises were derived from InsP3-sensitive Ca2+ stores. The concentration dependence of ACh-evoked increases in the total endothelial [Ca2+]i signal was explained by additional cells responding to ACh and an increase in [Ca2+]i for each cell. Significantly, InsP3-evoked [Ca2+]i was reduced in >83% of arteries (n = 6, p<0.01, one-way ANOVA with Tukey’s post hoc test) as intraluminal pressure was increased (60 to 110 to 160 mmHg). These results provide a new method to visualise and analyse endothelial [Ca2+]i and suggest that mechanical forces are recognised by the endothelium and translated into biological responses, at least in part, by changes in InsP3-mediated Ca2+ signalling.