Pain is frequently associated with a wide range of diseases and more people suffer from chronic pain than diabetes, heart diseases and cancer combined in the United States. Currently available therapeutic strategies employ non-narcotic and narcotic medications that have limitations due to adverse effects, tolerance, dependence and sometimes ineffectiveness of the therapy. Therefore, developing novel treatment strategies that alleviate pain without serious adverse effects or addictive potential are ongoing. Recent research and clinical investigations suggest the potentials of botulinum neurotoxin A (BTX-A) as an emerging alternative to treat certain modalities of pain including headache and migraine without serious adverse effects and dependence. In a clinical investigation, local injection of BTX-A has been shown to produce a faster and long-lasting relief from chronic pain associated with peripheral vascular diseases accompanied by increased tissue perfusion. Although BTX-A is effective in treating pain, the mechanisms underlying its analgesic action are not fully understood. One of the possibilities is that BTX-A may not reach the target site, especially if it is located intracellularly. In this study, we show using patch-clamp techniques that exposure of BTX-A to intracellular surface of the plasma membrane inhibits voltage-gated sodium channels (VGSCs) and voltage-gated calcium channels (VGCCs) in dissociated rat dorsal root ganglion (DRG) neurons. The firing frequency of action potentials decreased and the duration of action potential prolonged, reflecting the blockade of VGSCs. We propose that the BTX-A induced pain relief is partly due to a block of VGSCs and VGCCs and the vasodilatory effect of BTX-A is due to its action on VGCC.