Transcription factor p53, which was initially associated with cancer, has now emerged as an important regulator of inflammation and extracellular matrix homeostasis; two processes highly relevant to tendon repair. The goal of this study was to evaluate the effect of a p53 transactivation inhibitor, namely pifithrin-, on the pathophysiological sequence following collagenase-induced tendon injury. Administration of pifithrin- during the inflammatory phase reduced the accumulation of neutrophils and macrophages by 30% and 40%, respectively, on day 3 post-injury. Pifithrin- failed to reduce the percentage of apoptotic cells following collagenase injection but delayed functional recovery. In uninjured Achilles tendons, pifithrin- increased metalloprotease activity by 2.4-fold. Accordingly, pifithrin- reduced the collagen content in intact tendons as well as in injured tendons 7 days post trauma, when compared to placebo. The effect of pifithrin- on load to failure and stiffness was also evaluated. The administration of pifithrin- during the inflammatory phase did not significantly decrease the functional deficit 3 days post trauma. More importantly, load to failure and stiffness were significantly decreased in the pifithrin- group from day 7 to day 28 when compared to placebo. Overall, our results suggest that administration of pifithrin- alters the inflammatory process and delays tendon healing. The present findings also support the concept that p53 can regulate extracellular matrix homeostasis in vivo.