Voltage-gated sodium channel activity enhances the motility and oncogene expression of metastasic cancer cells that express a neonatal alternatively spliced form of the NaV1.5 isoform. We reported previously that FS50, a salivary protein from Xenopsylla cheopis, showed inhibitory activity against the NaV1.5 channel when assayed in HEK 293T cells and antiarrhythmia effects on rats and monkeys after induction of arrhythmia by BaCl2. This study aims to identify the effect of FS50 on voltage-gated sodium channel activity and the motility of MDA-MB-231 human breast cancer cells in vitro. NaV1.5 was abnormally expressed in the highly metastatic breast cancer cell line MDA-MB-231, but not in the MCF-7 cell line. FS50 significantly inhibited sodium current, migration, and invasion in a dose-dependent manner, but had no effect on the proliferation of MDA-MB-231 cells at the working concentrations (1.5-12 μmol/l) after a long-term treatment for 48 h. Meanwhile, FS50 decreased NaV1.5 mRNA expression without altering the total protein level in MDA-MB-231 cells. Correspondingly, the results also showed that MMP-9 activity and the ratio of MMP-9 mRNA to TIMP-1 mRNA were markedly decreased by FS50. Taken together, our findings highlighted for the first time an inhibitory effect of a salivary protein from a blood-feeding arthropod on breast cancer cells through the NaV1.5 channel. Furthermore, this study provided a new candidate leading molecule against antitumor cells expressing NaV1.5.