The role of topically applied mitomycin C in preventing postoperative perineural fibrosis was examined by gross anatomical dissection and histological analysis in rats. The sciatic nerve was exposed bilaterally in 24 Wistar adult male rats, and an abrasion injury was produced on the exposed surface of the biceps femoris muscle in all animals. In the experimental group, cotton pads soaked with mitomycin C (0.5 mg/ml) were placed around the nerves for 5 min, whereas cotton pads soaked with saline were applied to the control group. Four weeks after surgery, the neurolysis sites were evaluated by blinded surgical dissection. Perineural adhesions were graded using a numerical grading scheme. The scar tissue formation index was also calculated, and a grading was made according to the number of fibroblasts/fibrocytes counted around the epineurium in histological evaluation. Mitomycin C-treated nerves showed significantly less perineural adhesions than controls. Quantification of the dense connective tissue surrounding the nerves revealed a statistically significant reduction around nerves treated with mitomycin C, and the number of fibroblast/fibrocytes was also significantly reduced. Application of topical mitomycin C might be effective in preventing epineural scar formation after neurolysis of peripheral nerves.