The aim of this study was to investigate the roles of the cathepsin B cysteine protease and its endogenous inhibitor stefin A in laryngeal cancer. Immunohistochemistry was employed to detect the expression of cathepsin B and stefin A in 84 patients with laryngeal cancer, respectively. The protein expression of stefin A was negatively associated with lymphatic metastasis, recurrence of laryngeal cancer and the survival rate, which was not observed with cathepsin B protein expression. Both down-regulation of cathepsin B and up-regulation of stefin A in vitro significantly inhibited the migration, invasion and proliferation of laryngeal cancer cells, respectively. Our results strongly suggest that stefin A may be a potential predictor of laryngeal cancer and may be used in the molecular diagnosis and gene therapy of laryngeal cancer. Cathepsin B may be used as a promising therapeutic target in the treatment of laryngeal cancer.