This study aimed to investigate the effects of intra-articular botulinum toxin in preventing arthrofibrosis. Arthrofibrosis was induced in both stifle joints of 20 rabbits by transecting the anterior cruciate ligament under intramuscular anesthesia with ketamine and xylazine. Intra-articular toxin at a dose of 0.6 ml (50 unit) and physiologic saline solution (0.6 ml) were injected into the right and left stifle joints, respectively, 3 times with a 1-week interval between each injection. The rabbits were euthanized in the 12th week via high dose anesthesia to remove the stifle joint. The severity of adhesions was assessed, applying a universal scoring system. Also the stifle joints were histologically evaluated for fibrosis. With regards to severity of adhesion a significant reduction in the adhesion score was observed in the toxin-treated group in comparison to untreated controls with mean ± SE values of 0.2 ± 0.1 and 2.4 ± 0.2, respectively ( p < 0.01). The histological evaluation showed no significant fibroblast in the toxin-treated group versus dense fibers with mature fibroblasts in the control group. Our results suggest that botulinum toxin demonstrated efficacy in preventing adhesion after knee surgery and all the parameters monitored showed consistent statistically significant improvement.