We have shown previously that functional voltage-gated Na+ channels (VGSCs) are expressed in metastatic human breast cancer (BCa) in vitro and in vivo (Fraser et al. 2005). The predominant VGSC isotype is the TTX-resistant ‘neonatal’ splice variant of Nav1.5 (nNav1.5). Furthermore, TTX-resistant VGSCs can potentiate metastatic behaviour in MDA-MB-231 cells (Fraser et al. 2005). The aim of the present study was to ascertain the specific involvement of the nNav1.5 in the VGSC-dependent metastatic behaviour of MDA-MB-231 cells. All data are presented as mean ± S.E.M. Statistical significance was evaluated with Student’s t tests. A polyclonal antibody (NESO-pAb), raised against an external epitope on nNav1.5, reduced both VGSC current density (Chioni et al. 2005) and in vitro migration through 12 μm-pore Transwell filters. The reduction in migration was dose-dependent, reaching 38 ± 6% at 1 μg/ml (P < 0.001; n = 7). NESO-pAb also reduced Matrigel invasion by 49 ± 8% compared to control (P < 0.001; n = 7), but had no effect on proliferation. There was no effect on proliferation or invasion of PC-3M prostate cancer cells, where Nav1.7 is predominant (Diss et al. 2001). IgG and an unrelated polyclonal anti-laminin antibody had no effect on invasion or proliferation of either cell line. A small interfering RNA (siRNA) was used to specifically silence the nNav1.5 gene. A non-targeting siRNA was used as control. Five days post transfection, the siRNA reduced the level of nNav1.5 mRNA, quantified using real-time PCR normalised to cytochrome b5 reductase gene, by 91 ± 6%, compared to the control (P 20). Importantly, migration was suppressed by 43 ± 18% (P = 0.037; n = 5), and was not further reduced by TTX (10 μM). We conclude that nNav1.5 is indeed primarily responsible for the VGSC-induced enhancement of metastatic cell behaviour in MDA-MB-231 cells and that targeting nNav1.5 expression/activity may be useful in clinical management of metastatic BCa.