Pressure myography uses diameter measurements of isolated, pressurized arteries to assess smooth muscle and endothelial cell function. Here, we introduce the open-source pressure myograph system and analysis software, VasoTracker, which can be built in house for ~10% of the cost of commercial systems. We report on the development of the VasoTracker platform and demonstrate its use by assessing endothelium-dependent vasodilation. The complete VasoTracker system includes everything expected of a commercial pressure system (heated vessel chamber, temperature controller, pressure monitor, computer, camera, microscope, and acquisition/analysis software). The freely available VasoTracker software is a stand-alone interface for image acquisition and blood vessel diameter tracking that, although designed to work with VasoTracker hardware, can be used with any microscope/camera system supported by μManager. The software acquires, displays and records video images, and automatically tracks the diameter of blood vessels – all in real time. Temperature and pressure data, and user interventions (such as drug additions) can also be logged. VasoTracker enables the investigation of artery function using a wide array of protocols, such as those investigating vascular contraction, dilation, or the myogenic response. We used VasoTracker to investigate whether TRPV4 channels contribute to vascular relaxation induced by the muscarinic receptor agonist, acetylcholine (ACh). Second-order mesenteric arteries were isolated from male Sprague Dawley rats (10-12 week old; 250 – 300 g) euthanized by intraperitoneal injection with pentobarbital sodium (200 mg kg−1). Arteries were mounted on a VasoTracker pressure myograph system, pressurized to 70 mmHg, and pre-constricted using phenylephrine. The selective TRPV4 agonist, GSK1016790A (GSK; 20 nM; applied intraluminally, ~ 100 µl min-1) relaxed PE-constricted vessels (n = 5). These GSK-evoked relaxations were reversed by the selective TRPV4 channels antagonist, HC067047 (20 µM; 70.5 ± 6.1 % relaxation for control; 8.8 ± 2.1 % for HC067047; n = 5; p < 0.05; paired t-test). ACh also relaxed pre-constricted arteries (n = 5). However, ACh-evoked relaxations were unaffected by HC067047 (90.0 ± 5.5 % relaxation for control; 82.6 ± 8.1 % for HC067047; n = 5; p < 0.05; paired t-test). These data suggest that TRPV4 channels, whilst present in rat mesenteric arteries, do not contribute to ACh-evoked vasodilation. In releasing VasoTracker, we provide the vascular community with an inexpensive, transparent and fully customizable tool for studying blood vessel function. Details on how to build and use the system, and the VasoTracker software are freely available from the publicly accessible website, www.vasotracker.com. We hope that the open documentation will enable researchers to expand and enhance the platform for tailored vascular reactivity experiments.