The purpose of this study was to evaluate the role of TGF-β1 in regulating tendon extracellular matrix (ECM) after acute exercise. Wistar rats exercised (n=15) on a treadmill for four consecutive days (60 min/day) or maintained normal cage activity. After each exercise bout, the peritendinous space of each Achilles tendon was injected with a TGF-β1 receptor inhibitor or sham. Independent of group, tendons injected with inhibitor exhibited ~50% lower Smad 3 (Ser423/425) (p<0.05) and 2.5-fold greater ERK1/2 phosphorylation (p<0.05) when compared to sham (p<0.05). Injection of the inhibitor did not alter collagen content in either group (p>0.05). In exercised rats, hydroxylyslpyridinoline (HP) content and collagen III expression were lower (p<0.05) in tendons injected with inhibitor when compared to sham. In non-exercised rats, collagen I and lysyl oxidase (LOX) expression were lower (p<0.05) in tendons injected with inhibitor when compared to sham. Decorin expression was not altered by inhibitor in either group (p>0.05). Based on evaluation of hematoxylin and eosin (H&E) stained cross-sections, cell numbers were not altered by inhibitor treatment in either group (p>0.05). Evaluation of H&E stained sections revealed no effect of inhibitor on collagen fibril morphology. In contrast, scores for regional variation in cellularity decreased in exercised rats (p<0.05). No differences in fiber arrangement, structure, and nuclei form were noted in either group (p>0.05). Our findings suggest that TGF-1 signaling is necessary for the regulation of tendon cross-link formation as well as collagen and LOX gene transcription in an exercise-dependent manner.