BACKGROUND AND PURPOSE The mu-conopeptide family is defined by its ability to block voltage-gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new mu-conopeptide (mu-CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH mu-CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. mu-CnIIIC was also studied on HEK-293 cells expressing various rodent VGSCs and also on voltage-gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross-interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS Synthetic mu-CnIIIC decreased twitch tension in mouse hemidiaphragms (IC50= 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with mu-SIIIA, mu-SmIIIA and mu-PIIIA. mu-CnIIIC blocked NaV1.4 (IC50= 1.3 nM) and NaV1.2 channels in a long-lasting manner. Cardiac NaV1.5 and DRG-specific NaV1.8 channels were not blocked at 1 mu M. mu-CnIIIC also blocked the a3 beta 2 nAChR subtype (IC50= 450 nM) and, to a lesser extent, on the a7 and a4 beta 2 subtypes. Structure determination of mu-CnIIIC revealed some similarities to a-conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS mu-CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.