Nitric oxide (NO) has a variety of physiological roles, including acting as a key mediator in various phases of tendon healing, but its importance as a modulator of gene expression during tendon healing has not been well studied. The current study used microarray analysis to elucidate global gene expression after transfection with inducible nitric oxide synthase (iNOS) in tenocytes isolated from the injured rotator cuff tendons of human patients. We show that the expression of a wide range of genes is affected by NO, with many activated genes having known roles in healing. Of particular significance is that NOS overexpression stimulates the transcription and translation of a range of extracellular matrix genes important to the structure of connective tissues such as tendons, including collagen Ialpha1, collagen IIIalpha1, collagen IValpha5, biglycan, decorin, laminin, and matrix metalloproteinase 10 (MMP10). These genes were also shown to respond to stimulation by the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) in a dose-dependent manner. We further show that varying levels of NO significantly affect cellular adhesion in tenocytes, a critical process during tendon repair. These findings will be of use when optimizing the dose of NO delivery in further work investigating NO as potential treatment of tendon injuries.