Nitric oxide (NO) is a mediator involved in bone regeneration. We therefore examined the effect of the novel NO donor, S-nitroso human serum albumin (S-NO-HSA) on bone formation in a rabbit calvaria augmentation model. Circular grooves (8mm diameter, two per animal) were created by a trephine drill in the cortical bone of 40 rabbits and titanium caps were placed on the rabbit calvaria bone filled with a collagen sponge soaked with either 100μL S-NO-HSA (5%, 20%) or human albumin (5%, 20%). After 4 weeks the titanium hemispheres were subjected to histological and histomorphometric analysis. Bone formation and the volume of the residual collagen sponge were evaluated. S-NO-HSA treatment groups had a significantly higher volume of newly formed bone underneath the titanium hemispheres compared to the albumin control groups (5%: 15.5±4.0% versus 10.6±2.9%; P<0.05; 20%: 14.0±4.6% versus 6.0±3.8%; P<0.01). The volume of residual collagen sponge was also significantly lower in the S-NO-HSA groups compared to the control groups (5%: 0.4±0.5% versus 2.6±2.4%; P<0.05 and 20%: 1.5±2.7% versus 13.0±18.7%; P<0.01). This study demonstrates for the first time that S-NO-HSA promotes bone formation by slow NO release. Additionally, S-NO-HSA increases collagen sponge degradation.