Nitric oxide (NO) donors decrease intraocular pressure (IOP) by increasing aqueous outflow facility in the trabecular meshwork (TM) and/or Schlemms canal. However the cellular mechanisms are unknown. Cellular mechanisms known to regulate outflow facility include changes in cell volume and cellular contractility. In this study we investigated the effects of NO donors on outflow facility and the NO-induced effects on TM cell volume. We tested the involvement of soluble guanylate cyclase (sGC), cyclic GMP (cGMP), protein kinase G (PKG) and the BK_Ca channel using inhibitors and activators. Cell volume was measured using Calcein AM fluorescent dye, detected by confocal microscopy and quantified using NIH ImageJ software. An anterior segment organ perfusion system measured outflow facility. NO increased outflow facility in porcine eye anterior segment (0.4884 - 1.3956 µl/min/mmHg) over baseline (0.2373-0.5220 µl/min/mmHg) within 10 minutes of drug application. This NO-induced increases in outflow facility was inhibited by the the BK_Ca channel inhibitor, IBTX. Exposure of TM cells to NO resulted in 10% decrease in cell volume and these decreases were abolished by the sGC inhibitor, ODQ, and IBTX suggesting the involvement of sGC and K⁺ eflux respectively. The NO-induced decreases in cell volume were mimicked by 8-Br-cGMP and abolished by the protein kinase G (PKG) inhibitor, (RP)-8-Br-PET-cGMP-S suggesting the involvement cGMP and PKG. Additionally, the time course for the NO-induced decreases in TM cell volume correlated with NO-induced increases in outflow facility suggesting that the NO-induced alterations in cell volume may influence outflow facility.