Salivary adenoid cystic carcinoma (SACC) is a common type of salivary gland cancer. The poor long-term prognosis for patients with SACC is mainly due to local recurrence, perineural invasion (PNI) and distant metastasis. Notch signaling plays a critical role in determining cell fate such as proliferation, differentiation and apoptosis. Accumulating evidence indicates that aberrant Notch-4 expression has a tumor-promoting function in SACC. In the present study, we used lentiviral-mediated RNA interference (RNAi) targeted against Notch-4 to determine the effects of decreased levels of this protein in the human highly metastatic adenoid cystic carcinoma cell line ACC-M. Furthermore, the proliferative capability as well as the PNI potential of the treated cells were observed in vitro. Our studies demonstrated that RNAi directed against Notch-4 markedly decreased Notch-4 gene expression, resulting in the inhibition of cell proliferation, and G0/G1 to S phase arrest in ACC-M cells. Knockdown of Notch-4 also resulted in a decrease in the in vitro PNI activity in ACC-M cells. To conclude, RNAi targeting against Notch-4 induces the suppression of cell growth and inhibition of PNI in vitro in ACC-M cells. Notch-4 may play an important role in regulating proliferation and PNI activity of SACC.