Introduction: Osteoarthritis (OA) is the most common chronic musculoskeletal disorder. Nowadays, the role of synovial fluid mesenchymal stem cells (SF MSCs) is increasingly attributed to the treatment of Temporomandibular joint osteoarthritis. This study aimed to compare single versus multiple intra-articular injection of SFMSCs in terms of macroscopic, biochemical, and histological analysis. Material & methods: 18 young adult male 200–300 grams albino rats were used in the current study divided randomly into 3 equal groups. Group (A) comprised control in which OA was induced with no treatment, group (B) comprised MSCs single intra-articular injection group, and group (C) comprised MSCs multiple intra-articular injection group. Biochemical and histologic analysis were performed. Results: histological findings showed that both groups B, C showed regeneration of meniscus and retrodiscal tissues, but group C was superior in regeneration compared to group B. Biochemical analysis showed that TNF-α and IL-1B had the highest value in the control group, followed by the single injection group, while the lowest value was found in multiple injections group respectively. Conclusion: Multiple Intra-articular SF MSCs is a potential disease-modifying therapy for patients suffering from TMJ-OA.