Simple SummaryDespite the recent advances in the diagnosis and treatment of medullary thyroid cancer (MTC), it remains an understudied cancer type and continues to disproportionately contribute to thyroid-cancer-related mortality. In this manuscript, we report, for the first time, the overexpression of voltage-gated sodium channel subtype NaV1.7 in MTC cells and MTC patient samples, which is not expressed in normal thyroid cells and tissues. We establish the druggability of this channel by identifying a novel inhibitor (SV188) of this channel and investigate its mode of binding and ability to inhibit theINacurrent in NaV1.7. We also show thatSV188significantly inhibited the migration and invasion of aggressive MTC cells at doses lower than its cytotoxic concentration. Overall, our data suggest that the unique overexpression of NaV1.7 in MTC can be exploited for the discovery of novel small-molecule drugs to treat MTC metastasis.AbstractOur results from quantitative RT-PCR, Western blotting, immunohistochemistry, and the tissue microarray of medullary thyroid cancer (MTC) cell lines and patient specimens confirm that VGSC subtype NaV1.7 is uniquely expressed in aggressive MTC and not expressed in normal thyroid cells and tissues. We establish the druggability of NaV1.7 in MTC by identifying a novel inhibitor (SV188) and investigate its mode of binding and ability to inhibitINacurrent in NaV1.7. The whole-cell patch-clamp studies of theSV188in the NaV1.7 channels expressed in HEK-293 cells show thatSV188inhibited theINacurrent in NaV1.7 with an IC50value of 3.6 µM by a voltage- and use-dependent blockade mechanism, and the maximum inhibitory effect is observed when the channel is open.SV188inhibited the viability of MTC cell lines, MZ-CRC-1 and TT, with IC50values of 8.47 μM and 9.32 μM, respectively, and significantly inhibited the invasion of MZ-CRC-1 cells by 35% and 52% at 3 μM and 6 μM, respectively. In contrast,SV188had no effect on the invasion of TT cells derived from primary tumor, which have lower basal expression of NaV1.7. In addition,SV188at 3 μM significantly inhibited the migration of MZ-CRC-1 and TT cells by 27% and 57%, respectively.