Bupivacaine ranks the most potent and efficient drug among class I local anesthetics (LAs), but its high potential for toxic reactions severely prevent its use in clinical operations. Although bupivacaine-induced toxicity is mainly caused by substantial blockade of voltage-gated sodium channels (VGSCs), how these hydrophobic molecules interact with the receptor sites they bind with remains unclear. Nav1.5 is the dominant isoform of VGSCs expressed in cardiac myocytes, and dysfunction of Nav1.5 might be the cause of bupivacaine-triggered arrhythmia. Here, we investigate the effect of bupivacaine on Nav1.5 at clinical concentration range, aiming to illustrate the underlying molecular mechanisms. The electrophysiological measurements on Nav1.5 expressed in Xenopus oocytes showed that bupivacaine could induce a voltage- and concentration-dependent block on the peak of INa and the half-maximal inhibition dose (IC50) was 4.51 μM. Consistent with other LA drugs, bupivacaine could also induce a use-dependent block on Nav1.5 currents. The underlying mechanisms of this block may contribute to the fact that bupivacaine could not only dose-dependently affect the gating kinetics of Nav1.5 but also accelerate the development of open-state slow inactivation of Nav1.5. This study may extend our knowledge of the action of bupivacaine on cardiac sodium channels, and therefore, contribute to safer and more efficient clinical practice of bupivacaine.