Voltage-gated Na+ channels (Nav) regulate the excitability of sensory neurons and are potential targets for novel analgesics. The secreolytic ambroxol reduces pain-related behavior in rodents and alleviates pain in humans. With properties resembling those of local anesthetics, ambroxol has been reported to block Na+ currents in sensory neurons with a preference for tetrodotoxin-resistant (TTXr) Na+ currents encoded by Nav1.8. However, the molecular determinants for ambroxol-induced block of Na+ channels and a preferential block of Nav1.8 opposed to tetrodotoxin-sensitive (TTXs) Nav α-subunits have not been studied in detail. By means of whole-cell voltage clamp recordings, we studied the effects of ambroxol and local anesthetics on the recombinant TTXr subunit Nav1.8, on TTXs Nav α-subunits and on mutants of Nav1.4 that are insensitive to local anesthetics. Tonic and use-dependent block by ambroxol was strongly alleviated in local anesthetic-insensitive Nav1.4 mutants. Use-dependent block, but not tonic block was significantly stronger on Nav1.8 than on TTXs channels. The TTXs subunit Nav1.3 displayed the least degree of use-dependent block by ambroxol. The local anesthetics mepivacaine and S(-)-bupivacaine also blocked Nav1.8 and TTXs channels differentially. While mepivacaine displayed a preferential use-dependent block of Nav1.8, S(-)-bupivacaine displayed a preference for TTXs Na+ channels. Our data show that ambroxol acts as a typical local anesthetic on Na+ channels interacting with specific residues in the S6 segments. This property probably meditates the analgesic effect of ambroxol. Ambroxol preferentially blocks Nav1.8, however shares this property with established local anesthetics like mepivacaine.