The purpose of this study is to investigate the specific innervations on “Weizhong” (BL40) and “Huantiao” (GB30) by using a dual neural tracing technique. After Alexa Fluor 488 and 594 conjugates of cholera toxin subunit B (AF488/594-CTB) were, respectively, injected into BL40 and GB30 in the same rat, the labeled sensory and motor neurons were examined in the rat’s dorsal root ganglia (DRGs) and spinal cord at thoracic (T) and lumbar (L) segments with a laser scanning confocal microscope. In the cases of BL40 injection, AF488-CTB labeled sensory and motor neurons were located in L2–6 DRGs and on the mediolateral part of spinal ventral horn from L3 to L5 segments, respectively. By contrast, in the cases of GB30 injection, AF594-CTB labeled sensory and motor neurons were distributed in T13-L6 DRGs and on the anterolateral part of spinal ventral horn from L1 to L5 segments, respectively. These results indicate that the sensory and motor neurons associated with BL40 and GB30 are located in different spinal segments and regions in the nervous system, providing the neuroanatomical evidence to serve the specificity of acupoints.