Posttraumatic osteoarthritis (PTOA) is a common sequela to anterior cruciate ligament (ACL) injury in humans and dogs. Identification of conserved targets between species may hasten biomarker identification and therapeutic development. To identify differentially regulated synovial fluid (SF) proteins in ACL injury, SF samples were collected from knees with ACL injury or control knees (n = 8 per species) in human and dog patients. The SF proteome was evaluated using nano-scale reverse-phase chromatography and tandem mass spectrometry. Associations between proteins and ACL injury were analyzed in both species. Immunoassays were used to validate key proteins identified via mass spectrometry, including periostin, α-2-macroglublin, and lubricin, in additional SF samples (dog:​ n = 57 ACL, n = 31 controls;​ human:​ n = 20 ACL, n = 15 advanced OA) and plasma samples (human:​ n = 18 ACL, n = 15 advanced OA, and n = 12 controls). Periostin was the most upregulated SF protein (log2 fold change) in both dogs (2.6) and humans (3.5) with ACL injury, followed by complement C1q (2.3) and α-2-macroglobulin (1.8) in dogs and fibrinogen (1.1) and α-2-macroglobulin (1.1) in humans. Most downregulated proteins included serum amyloid A1 (−1.9) and aggrecan (−1.8) in dogs and carbonic anhydrase 2 (−3.2) and hemoglobin subunit β (−2.8) in humans. Approximately 60% of proteins detected were shared between species, and immunoregulatory and macromolecular transport proteins were the most common families. Findings in both species support further investigation into periostin as a potential biomarker or therapeutic target for joint injury and the use of spontaneous ACL injury in dogs as a translational large animal model for human ACL injury.