We investigated the hypothesis that hindlimb unloading inhibits healing in fibrous connective tissue such as ligament. Male rats were assigned to 3- and 7-wk treatment groups with three subgroups each: sham control, ambulatory healing, and hindlimb-suspended healing. Ambulatory and suspended animals underwent surgical rupture of their medial collateral ligaments, whereas sham surgeries were performed on control animals. After 3 or 7 wk, mechanical and/or morphological properties were measured in ligament, muscle, and bone. During mechanical testing, most suspended ligaments failed in the scar region, indicating the greatest impairment was to ligament and not to bone-ligament insertion. Ligament testing revealed significant reductions in maximum force, ultimate stress, elastic modulus, and low-load properties in suspended animals. In addition, femoral mineral density, femoral strength, gastrocnemius mass, and tibialis anterior mass were significantly reduced. Microscopy revealed abnormal scar formation and cell distribution in suspended ligaments with extracellular matrix discontinuities and voids between misaligned, but well-formed, collagen fiber bundles. Hence, stress levels from ambulation appear unnecessary for formation of fiber bundles yet required for collagen to form structurally competent continuous fibers. Results support our hypothesis that hindlimb unloading impairs healing of fibrous connective tissue. In addition, this study provides compelling morphological evidence explaining the altered structure-function relationship in load-deprived healing connective tissue.