Objective: Increasing evidence from acupuncture research suggests that the nervous system corresponds closely with classical acupuncture points. The aim of this research was to provide neuroanatomical evidence for revealing the innervated properties of different acupuncture points through comparing the sensory and motor pathways associated with Hegu (LI 4) and Taichong (LR 3) in rat extremities. Materials and Methods: Cholera toxin subunit B (CTB) was injected into LI 4 and LR 3 in different rats, and CTB neural labeling was examined using fluorescent immunohistochemistry and observed under fluorescent microscopy in the corresponding areas from the peripheral nervous system to the central nervous system, including the dorsal root ganglia (DRG), spinal cord, and brainstem. Results: When LI 4 was injected with CTB, CTB-labeled sensory neurons ranged from C5 to T1 DRG, and their transganglionic axons terminated in the C5 to C8 spinal dorsal horn as far as the cuneate nucleus, while labeled motor neurons were located in the C7 to T1 spinal ventral horn. In contrast, similar neural labeling was observed for LR 3 CTB injection, with an orderly arrangement in the L3 to L5 DRG, L3 to L5 spinal dorsal horn, gracile nucleus, and L4 to L6 spinal ventral horn. Conclusions: The present results provide further evidence to aid understanding of the differential innervation of acupuncture points LI 4 and LR 3. This innervation establishes its connection with the nervous system in a distinct segmental and regional pattern through the spinal sensory and motor pathways.