Tendon exhibits nonlinear stress–strain behavior that may be partly due to movement of collagen fibers through the extracellular matrix. While a few techniques have been developed to evaluate the fiber architecture of other soft tissues, the organizational behavior of tendon under load has not been determined. The supraspinatus tendon (SST) of the rotator cuff is of particular interest for investigation due to its complex mechanical environment and corresponding inhomogeneity. In addition, SST injury occurs frequently with limited success in treatment strategies, illustrating the need for a better understanding of SST properties. Therefore, the objective of this study was to quantitatively evaluate the inhomogeneous tensile mechanical properties, fiber organization, and fiber realignment under load of human SST utilizing a novel polarized light technique. Fiber distributions were found to become more aligned under load, particularly during the low stiffness toe-region, suggesting that fiber realignment may be partly responsible for observed nonlinear behavior. Fiber alignment was found to correlate significantly with mechanical parameters, providing evidence for strong structure–function relationships in tendon. Human SST exhibits complex, inhomogeneous mechanical properties and fiber distributions, perhaps due to its complex loading environment. Surprisingly, histological grade of degeneration did not correlate with mechanical properties.