To explore the therapeutic efficacy of L-glutamine (L-Gln) on pathological progression and clinical symptoms of osteoarthritis (OA), and compare with glucosamine sulfate (GS), and celecoxib (CXB). Rats were administered sodium chloride, L-Gln, GS, or CXB via gavage for eight weeks starting from the fifth week after sham operation or Anterior Cruciate Ligament Transection (ACLT) + Medial Meniscectomy (MMx). Then the severity of knee OA in rats was evaluated by serological analysis, histological examination and imaging examination. In addition, patients with mild primary OA were administered L-Gln, GS, or CXB orally for 12 weeks in accordance with the randomization principle. The efficacy end points were the change from baseline to week 24 in the pain and physical function subscale scores of the Western Ontario and McMaster Universities OA Index (WOMAC), and Lequesne score. Treatment with L-Gln alleviated the increased concentration of serum cartilage degradation markers caused by OA in rats. Histological tests showed improvement in knee joint cartilage destruction after treatment. Three-dimensional CT scans and reconstructions revealed a reduction in osteophyte formation and subchondral bone loss. L-glutamine performed as well as or better than glucosamine sulfate and celecoxib in all comparative measures among the three treatment groups. In clinical trials, the WOMAC pain and physical function subscale scores, as well as the Lequesne score, decreased from baseline in all three patient groups during follow-up, with no significant differences observed between the groups. Our research indicates that L-Gln is comparable to GS and CXB in improving the pathological progression and clinical efficacy of OA, which makes it a promising drug for the treatment of osteoarthritis.