In this work, two P-superfamily conotoxins, lt9a and lt9b, were purified and characterized from the crude venom of Conus litteratus. The amino acid sequences of lt9a and lt9b were determined by the Edman degradation method. It has been suggested that both lt9a and lt9b are produced from the precursor encoded by the gene Lt9.1. During the conotoxin maturation process, different post-translational modifications occurred between lt9a and lt9b. Conotoxin lt9b was predicted to have two prolines that underwent hydroxylation and one glutamate that underwent carboxylation, while lt9a had no hydroxyproline and carboxyglutamate residue. The calculated mass weights of two P-superfamily conotoxins with three proposed disulfide bonds were confirmed by matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrometry after considering corresponding post-translational modifications. These two conotoxins showed different effects on tetrodotoxin-sensitive sodium currents. Conotoxin lt9a (300 nM) resulted in marked slowing of the tetrodotoxin-sensitive sodium current decay, a notable increase in the peak current, and an alteration in reversal potential. However, lt9b inhibits tetrodotoxin-sensitive sodium currents, and the inhibition showed a concentration-dependent with the half maximal inhibitory concentration (IC50) value of 504.04 nM, but there were no change in the activation and inactivation kinetics of currents. To the best of our knowledge, this is the first investigation of two P-superfamily conotoxins identified to act on voltage-sensitive sodium channels with different modifications.