Activity of the voltage-gated Nav1.5 sodium channel has been reported to be involved in cell proliferation, cancer invasion and gene expression. In addition, eicosapentaenoic acid (EPA) has recently been suggested to inhibit ovarian cancer cell growth and suppress tumor metastasis. The present study aimed to explore the association between EPA, the Nav1.5 sodium channel and ovarian cancer cells. Using patch-clamp technique and RNA interference approaches, sodium currents were recorded in epithelial ovarian cancer cells, and it was confirmed that the Nav1.5 channel carried the sodium currents. Furthermore, EPA effectively inhibited sodium currents in a dose-dependent manner, shifted the steady-state inactivation curve of sodium currents to the hyperpolarizing direction and reduced sodium window currents. In addition, EPA induced a shift in the inactivation curve in a dose-dependent manner. Inhibition of the sodium channel, either by EPA or by Nav1.5 knockdown, attenuated ovarian cancer cell migration and proliferation. To the best of our knowledge, the present study is the first to conduct sodium current recording in ovarian cancer cells, and revealed that EPA may inhibit Nav1.5-mediated ovarian cancer cell migration and growth. These findings not only present a potential prognostic biomarker for ovarian cancer, but also provide a strategy towards the development of novel pharmacological treatments for patients with ovarian cancer.