This study compared the use of a diet enriched with fish oil with a control diet on synovial fluid matrix metalloproteinases (MMP), tissue inhibitors of MMP-2 (TIMP-2) and urokinase plasminogen activator (uPA) in dogs with acute cranial cruciate ligament (CCL) injury. Dogs were fed either the control or study diet from 1 week before surgery to 56 days post surgery and plasma and synovial fluid (from both the injured and the healthy knee joints) were collected and analysed 7 days before surgery, on surgery day itself and 7, 14, 28 and 56 days post surgery. Results showed that plasma total EPA and DHA were significantly elevated, and plasma total arachidonic acid (AA) was significantly reduced by the fish oil diet. Whilst there were no significant changes in dogs fed the fish oil diet in the surgical joint in MMP expression or activity, TIMP-2 expression or uPA activity at any time during the study; beneficial changes were observed in the synovial fluid of the healthy knee joint. This suggests that dietary fish oil may bring about positive effects on synovial fluid MMP and TIMP-2 equilibrium in the uninjured stifle of dogs with CCL injury.