The mechanisms of flow-induced vascular remodeling are poorly understood, especially in the coronary microcirculation. We hypothesized that application of flow in small coronary arteries in organoid culture would cause a nitric oxide (NO)-mediated dilation and inhibit inward remodeling. We developed an organoid culture setup to drive a flow through cannulated arterioles at constant luminal pressure via a pressure gradient between the pipettes. Subepicardial porcine coronary arterioles with diameter at full dilation and 60 mmHg ( D₀) of 168 ± 10 (SE) μm were cannulated. Vessels treated with N^ω-nitro-l-arginine (l-NNA) to block NO production and untreated vessels were pressurized at 60 mmHg for 3 days with and without flow. Endothelium-dependent dilation to 10⁻⁷M bradykinin was preserved in all groups. Tone was significantly less in vessels cultured under flow conditions in the last half of the culture period. Untreated and l-NNA-treated vessels regulated their diameter to yield shear stresses of 10.3 ± 2.1 and 14.0 ± 2.4 (SE) dyn/cm², respectively (not significantly different). Without l-NNA, passive pressure-diameter curves at the end of the culture period revealed inward remodeling in the control group [to 92.3 ± 1.3% of D₀(SE)] and no remodeling in the vessels cultured under flow conditions (100.2 ± 1.3% of D₀); with l-NNA, the group subjected to flow showed inward remodeling (92.1 ± 2.5% of D₀). We conclude that pressurized coronary resistance arteries could be maintained in culture for several days with flow. Vessels cultured under flow conditions remained more dilated when NO synthesis was blocked. Inward remodeling occurred in vessels cultured under no-flow conditions and was inhibited by flow-dependent NO synthesis.