Periostin is an extracellular matrix protein involved in the regulation of intercellular adhesion. The present study investigated the in vivo tumor suppressor function of periostin in a mouse orthotopic model of bladder cancer. Retroviral vectors were used to transfect human bladder cancer UMUC-3 cell line with periostin. Bladders of nude mice that were transurethrally instilled with periostin-expressing UMUC-3 cells were revealed to weigh less compared with bladders instilled with vector control cells. In total, five (83.3%) of six vector control UMUC-3 bladder tumors exhibited histological evidence of muscle invasion. However, none of the five periostin-expressing UMUC-3 bladder tumors revealed muscle invasion. Thick edematous lesions were present in the submucosa of periostin-expressing UMUC-3 bladder tumors. The expression of periostin also suppressed in vitro cell invasiveness of UMUC-3 cells without affecting cellular proliferation. The level of phosphorylation of phosphoinositide-dependent kinase-1 (PDK1), protein kinase B (Akt) and S6 ribosomal protein, a downstream protein of mammalian target of rapamycin (mTOR) was decreased in periostin-expressing UMUC-3 cells compared with vector control cells. Treatment with 100 ng/ml recombinant human periostin protein also suppressed cell invasiveness and phosphorylation of PDK1, Akt and S6 in UMUC-3 cells, consistent with results using periostin-expressing UMUC-3 cells. Treatment with PDK1, Akt and mTOR inhibitors significantly suppressed UMUC-3 cell invasiveness. These results demonstrate that periostin suppresses in vivo and in vitro invasiveness of bladder cancer via the PDK1/Akt/mTOR signaling pathway. Periostin may be useful as a potent chemotherapeutic agent by suppressing bladder cancer invasiveness.