To determine if inducible nitric oxide synthase (iNOS) gene could affect Achilles tendon healing using iNOS gene knockout mice. 21 iNOS knockout (iNOS(-/-)) mice and 8 of the wild type (iNOS(+/+)) mice were utilized in this study. Group 1: iNOS(+/+) mice (n = 8), group 2: iNOS(-/-) mice (n = 11) and group 3: iNOS(-/-) with a NOS inhibitor, (aminoguanidine, 500 mg/kg/day, via an intraperitoneal mini-osmotic pump for 7 days, n = 10). The right Achilles tendon was transected in all mice and harvested on day 7 for cross-sectional area and biomechanical properties. Serum nitrate concentration of the mice was measured by gas chromatography mass spectrometry (GC/MS). A significant reduction in cross-sectional area of the healing Achilles tendon was observed in group 3 mice compared to group 2 mice (p < 0.01). The serum nitrate concentration in both group 2 and group 3 mice was lower than that in group 1 mice (p < 0.01) iNOS gene deletion and inhibition of NOS did not affect the biomechanical properties of the healing tendons. iNOS gene is not solely responsible for the beneficial effects of nitric oxide (NO) on tendon healing.