Hainantoxin-III and hainantoxin-IV, isolated from the venom of the Chinese bird spider Seleconosmia hainana, are neurotoxic peptides composed of 33–35 residues with three disulfide bonds. Using whole-cell patch-clamp technique, we investigated their action on ionic channels of adult rat dorsal root ganglion neurons. It was found that the two toxins did not affect Ca2+channels (both high voltage activated and low voltage activated types) nor tetrodotoxin-resistant voltage-gated Na+ channels (VGSCs). However, hainantoxin-III and hainantoxin-IV strongly depressed the amplitude of tetrodotoxin-sensitive Na+ currents with IC50 values of 1.1 and 44.6nM, respectively. Both hainantoxin-III (1nM) and hainantoxin-IV (50nM) caused a hyperpolarizing shift of about 10mV in the voltage midpoint of steady-state Na+ channel inactivation, but they showed difference in the reprime kinetics of VGSCs: hainantoxin-III significantly decreased the recovery rate from inactivation at a prepulse potential of 80mV while hainantoxin-IV did not do. It is interesting to note that similar to huwentoxin-IV, the two hainantoxins did not affect the activation and inactivation kinetics of Na+ currents and at a concentration of 1AM they completely inhibited the slowing inactivation currents induced by BMK-I (toxin I from the scorpion Buthus martensi Karsch), a scorpion a-like toxin.The results indicate that hainantoxin-III and hainantoxin-IV are novel spider toxins and affect the mammal neural Na+ channels through a mechanism quite different from other spider toxins targeting the neural receptor site 3, such as y-aractoxins and A-agatoxins.