A small library of synthetic (-)-palmyrolide A diastereomers, analogues, and acyclic precursors have been examined with respect to their interaction with voltage-gated sodium channels (VGSCs). Toward this goal, the ability of (-)-palmyrolide A and analogues to antagonize veratridine-stimulated Na+ influx in primary cultures of mouse cerebrocortical neurons was assessed. We found that synthetic (-)-palmyrolide A and its enantiomer functioned as VGSC antagonists to block veratridine-induced sodium influx. A detailed NMR and computational analysis of four diastereomers revealed that none had the same combination of shape and electrostatic potential as exhibited by natural (-)-palmyrolide A. These data indicate that the relative configuration about the tert-butyl and methyl substituents appears to be a prerequisite for biological function. Additional testing revealed that the enamide double bond was not necessary for blocking veratridine-induced sodium influx, whereas the acyclic analogues and other macrolide diastereomers tested were inactive as inhibitors of VGSCs, suggesting that the intact macrolide was required.