Purpose: To correlate outflow function and outflow tract vessel diameter changes induced by nitric oxide (NO). Methods: In a porcine anterior segment perfusion model, the effects of a nitric oxide donor (100 µM DETA-NO) on outflow facility were compared to controls (n=8 per group) with trabecular meshwork (TM) and after circumferential ab interno trabeculectomy (AIT). Outflow structures were assessed with spectral domain optical coherence tomography (SD-OCT) before and after NO, or an NO synthase inhibitor (100 µM L-NAME) and the vasoconstrictor, endothelin-1 (100 pg/mL ET-1). Scans were processed with a custom macro script and aligned for automated reslicing and quantification of cross-sectional outflow tract areas (CSA). Results: The facility increased after DETA-NO (0.189±0.081 μL/min·mmHg, p=0.034) and AIT (0.251±0.094 μL/min·mmHg, p=0.009), respectively. Even after AIT, DETA-NO increased the facility by 61.5% (0.190±0.074 μL/min·mmHg, p=0.023) and CSA by 13.9% (p<0.001). L-NAME + ET-1 decreased CSA by -8.6% (p<0.001). NO increased the diameter of focal constrictions 5.0±3.8 fold. Conclusions: NO can dilate vessels of the distal outflow tract and increase outflow facility in a TM-independent fashion. There are short, focally constricting vessel sections that display large diameter changes and may have a substantial impact on outflow.