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Background Phentolamine is a non-selective competitive α-adrenergic antagonist clinically used to treat different vascular-related diseases. Since recent data suggest an antagonistic effect of phentolamine on voltage-gated sodium channels (VGSCs), further electrophysiological analysis is essential to understand its effects on peripheral sensory nerves.Methods We examined the effects of phentolamine on the amplitude of the stimulus-evoked compound action potential (CAP) of A- and C-type nerve fibers derived from mice, pigs, and humans compared with the effects of lidocaine. To explore potential mechanisms of phentolamine action, we performed single nerve fiber recordings using skin-nerve preparations from wild-type and Na V1.8/NaV1.9 double knockout mice, along with manual and automated whole-cell patch-clamp electrophysiology on ND7/23 or HEK293 cells with heterologously expressed VGSCs.Results Phentolamine decreased CAP amplitudes in a concentration-dependent manner, with significantly lower concentrations needed to affect C-fibers compared with A-fibers. Co-application of the α-adrenergic agonist (R)-(-)-phenylephrine did not alter this effect, suggesting that α-adrenergic receptors do not mediate phentolamine’s action in this case. Phentolamine (100 µM) inhibited the electrically evoked action potentials (AP) in the majority of single-unit cutaneous C-fibers, while the same concentration did not provoke AP extinction in A-fibers. C-fibers from Na V1.8/NaV1.9 double knockout mice were significantly less sensitive to phentolamine than those from wild types. In patch-clamp experiments, phentolamine concentration-dependently blocked VGSCs, whereas NaV1.8 showed the highest sensitivity (IC50=10 µM).Conclusions Suppressing AP conduction and inhibiting VGSCs, phentolamine exhibits features reminiscent of local anesthetics, but with a stronger effect on C-fibers, which may be attributed to its stronger action on tetrodotoxin-resistant VGSCs. However, due to its markedly higher affinity for α-adrenergic receptors compared with VGSCs, systemic administration of phentolamine is limited by its adrenergic side effects. These findings suggest that phentolamine could be useful for exploring C-fiber function and provide a basis for the development of more selective and potent antinociceptive agents.