PURPOSE. There is a time-course correlation between nitric oxide (NO)-induced decreases in trabecular meshwork (TM) cell volume and NO-induced increases in outflow facility. The Schlemm's canal (SC) cells may also provide resistance to aqueous humor outflow; therefore, this study tests the involvement of the nitric oxide synthase (NOS) and NO signaling pathway and the BK Ca-channel in mediating SC cell volume decreases. METHODS. Cell volume was measured in low-passage human SC cells using calcein AM fluorescent dye; images were captured with a confocal microscope, and data were quantified using NIH ImageJ software. RESULTS. Inhibition of endogenous NOS resulted in a 7% increase in SC cell volume. Exposure of SC cells to DETA-NO resulted in a 12% to 16% decrease in cell volume that was abolished by the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (5 μM), the protein kinase G (PKG) inhibitor (RP)-8-Br-PET-cGMP-S (50 μM), and the high-conductance calcium-activated potassium channel (BK Ca channel) inhibitor iberiotoxin (50 nM). Hypertonic media significantly decreased SC cell volume by 14%, whereas hypotonic media significantly increased cell volume by 11.2%. CONCLUSIONS. These data suggest that endogenous NOS regulates steady state cell volume and the involvement of the NOS/NO/sGC/cGMP/PKG pathway and the BK Ca-channel in mediating NO-induced reductions in SC cell volume. These decreases in cell volume correlated with the time-course for NO-induced increases in outflow facility, suggesting that the NO-induced reduction in SC cell volume may also influence outflow facility.