Previous data from space flight studies indicate that injured muscle and bone heal slowly and abnormally compared to ground controls, strongly suggesting that ligaments or tendons may not repair optimally as well. Thus, the objective of this study is to investigate the biochemical and molecular gene expression of the collagen extracellular matrix in response to medial collateral ligament (MCL) injury repair in hindlimb unloaded (HLU) rodents. Male rats were assigned to 3-week and 7-week treatment groups with three subgroups each: sham control, ambulatory healing (Amb-healing) and hindlimb unloaded (HLU-healing) groups. Ambulatory and HLU animals underwent bilateral surgical transection of their medial collateral ligaments, while control animals were subjected to sham surgeries. All surgeries were performed under isoflurane anesthesia. After 3 weeks or 7 weeks of hindlimb unloading, rats were euthanized and MCLs were surgically isolated and prepared for molecular or biochemical analyses. Hydroxyproline concentration and hydroxylysylpyridinoline (HP) collagen cross-link contents were measured by HPLC and showed a substantial decrement in surgical groups. MCL tissue cellularity, quantified by DNA content, remained significantly elevated in all HLU-healing groups versus Amb-healing groups. MCL gene expression of Col1A2, Col3A1, Col5A1, fibronectin, decorin, biglycan, lysyl oxidase, MMP-2 and TIMP-1, measured by real time Q-PCR, demonstrated a differential expression in the HLU-healing groups compared to Amb-healing groups at both the 3 and 7 week time points. Together, these data suggest that HLU impacts dense fibrous connective tissue wound healing and confirms previous morphological and biomechanical data that HLU inhibits the ligament repair processes.