Objective Osteoarthritis (OA) is a major cause of musculoskeletal pain and disability worldwide. The investigation of disease-modifying treatment options for OA has become an important aspect of orthopedic care. To assess the effect of intra-articular and oral glucosamine sulfate (GS) versus placebo on osteoarthritis in a canine model. Materials In this randomized, placebo-controlled, double-blinded study, OA was induced by anterior cruciate ligament transection (ACLT) according to the Pond-Nuki model in 32 canines. All canines were allocated into 4 treatment subgroups with treatment administered for 8 weeks: GS (400 mg) intra-articular, placebo intra-articular, GS (200 mg/kg body weight) oral, and placebo oral. The contralateral nonoperated stifle (knee) served as control. After 8 weeks, the medial and lateral femoral condyles, the medial and lateral tibial plateau and patella were histologically examined and anatomic changes quantified by light microscopy using the modified Mankin score. Results After 8 weeks, mean Mankin score values significantly (P < 0.002) decreased in the intra-articular GS group (8.1; range 7.9-8.8) compared with the intra-articular placebo group (13.9; range 11.6-15.9) and again significantly (P < 0.002) in the oral GS group (12.1; range 9.9-12.7) compared with the oral placebo group (15.1; range 12.5-17.0). Mean Mankin score values were significantly (P < 0.002) lower in the intra-articular GS group compared with the oral GS group. Conclusion Both, intra-articular and oral administered GS significantly reduced histological signs of OA in the Pond-Nuki model, with the intra-articular application being more effective compared to oral administration.