The cardiac sodium channel Nav1.5 is a key contributor to the cardiac action potential, and dysregulations in Nav1.5 can lead to cardiac arrhythmias. Nav1.5 is a target of numerous antiarrhythmic drugs (AADs). Previous studies identified the protein 14-3-3 as a regulator of Nav1.5 biophysical coupling. Inhibition of 14-3-3 can remove the Nav1.5 functional coupling and has been shown to inhibit the dominant-negative effect of Brugada syndrome mutations. However, it is unknown whether the coupling regulation is involved with AADs’ modulation of Nav1.5. Indeed, AADs could reveal important structural and functional information about Nav1.5 coupling. Here, we investigated the modulation of Nav1.5 by four classic AADs, quinidine, lidocaine, mexiletine, and flecainide, in the presence of 14-3-3 inhibition. The experiments were carried out by high-throughput patch-clamp experiments in an HEK293 Nav1.5 stable cell line. We found that 14-3-3 inhibition can enhance acute block by quinidine, whereas the block by other drugs was not affected. We also saw changes in the use- and dose-dependency of quinidine, lidocaine, and mexiletine when inhibiting 14-3-3. Inhibiting 14-3-3 also shifted the channel activation toward hyperpolarized voltages in the presence of the four drugs studied and slowed the recovery of inactivation in the presence of quinidine. Our results demonstrated that the protein 14-3-3 and Nav1.5 coupling could impact the effects of AADs. Therefore, 14-3-3 and Nav1.5 coupling are new mechanisms to consider in the development of drugs targeting Nav1.5.