Objective To study the synthesis, identification and activity of three disulfide bond isomers of μ-CnIIIC. Methods The linear peptides of μ-CnIIIC-Ⅰ, μ-CnIIIC-Ⅱ and μ-CnIIIC-Ⅲ were synthesized by solid phase peptide synthesis (SPPS). Three-fold peptide isomers (μ-CnIIIC-Ⅰ, μ-CnIIIC-Ⅱ and μ-CnIIIC-Ⅲ) were obtained by oxidative folding of the linear peptides, which were characterized by mass spectrometry and purified by HPLC. The cytotoxicity of the three isomers was tested by MTT method. The inhibition effect of three isomers on NaV1.2 was tested by using double electrode voltage patch clamp technique. Results Mass spectrometry characterization results showed that μ-CnIIIC-Ⅰ, μ-CnIIIC-Ⅱ and μ-CnIIIC-Ⅲ were successfully synthesized. When the concentration of three isomers of μ-CnIIIC was in the range of 50 μmol/L, the cell survival rate was above 75% and no hemolysis occurred. When the concentration of μ-CnIIIC isomer was 20 μmol/L, the inhibition rates of μ-CnIIIC-Ⅰ, μ-CnIIIC-Ⅱ and μ-CnIIIC-Ⅲ on sodium channel were 23.64%, 52.43% and 81.21%, respectively. The average scores of skin irritation test were 0.107, 0.179 and 0.286 per animal per day. Conclusion The μ-CnIIIC isomers of conopeptides have good biosafety. The μ-CnIIIC isomers with disulfide bonds of Ⅰ-Ⅳ, Ⅱ-Ⅴ and Ⅲ-Ⅵ have better selectivity to sodium channels, stronger inhibitory effect and better biological activity.