The molecular features of parotid gland cancer (PGC) is not fully understood enough to develop an effective drug therapy because of the rarity. Since many human cancers in which tropomyosin receptor kinase B (TRKB) is highly expressed have poor prognosis, we here investigated the signaling by TRKB and the ligand brain-derived neurotrophic factor (BDNF) called the BDNF/TRKB pathway, in PGC cells using clinical specimens. In our primary culture system of patient-derived PGC cells and the cancer-associated fibroblasts (CAFs), the PGC cells co-cultured with CAFs showed a significant upregulation of BDNF and epithelial-mesenchymal transition. Additionally, the similar results were observed in PGC cells treated with conditioned medium from co-culture of PGC cells with CAFs. Moreover, TRK inhibitors suppressed BDNF-induced cell migration in PGC cells. In immunohistochemical and clinicopathological analyses of tumors from PGC patients, BDNF and TRKB were highly expressed in both tumor cells and stromal cells such as CAFs, and the expression level of TRKB in the PGC cells was significantly correlated with aggressive features including vascular invasion, nodal metastasis, and poor prognosis. Taken together, these data suggest that the BDNF/TRKB pathway may regulate PGC cell aggressiveness via cross-talk with CAFs, and could be a therapeutic target for PGC harboring invasive and metastatic features.