Periostin (PN), a secreted adhesion-related protein expressed in the periosteum and periodontal ligaments, acts as a critical regulator of the formation and maintenance of bone and teeth, and also plays an important role in tumorigenesis. Although PN is highly expressed in various types of human cancers, its function is still unclear. In this study, we focused on the exon 17 region of PN, which is alternatively spliced out. To investigate the function of full-length PN with exon 17, we produced a neutralizing antibody (PN1-Ab) against the peptide encoded by exon 17. In vivo, administration of PN1-Ab significantly inhibited the growth of primary tumors as well as metastatic tumors, associated with prevention of bone destruction, resulting in increased survival of mice. Consistent with in vivo data, the present in vitro study demonstrated that addition of full-length PN significantly inhibited cell adhesion and detached adherent cells, while PN1-Ab inhibited the action of PN in a dose-dependent manner. In addition, PN1-Ab significantly inhibited the proliferation, migration and invasion of 4T1 mouse breast cancer cells, which produced PN. Interestingly, PN1-Ab also inhibited the differentiation of osteoclasts. Overall, the present study demonstrated that PN plays a pivotal role in the progression and metastasis of breast cancer. Since administration of PN1-Ab prolonged cell survival through inhibition of the progression and metastasis of 4T1 cells, further development of the PN1-Ab such as generation of a humanized antibody may provide a new therapeutic agent against breast cancer.