Objective: A new retrograde neuron-tracing technique with microspheres was used to explore the possible innervation of calcitonin gene-related peptide (CGRP)-immunolabeled vestibular afferent neurons in the vestibular efferent immunolabeled nucleus in the brainstem. Methods: 0.1 µl of 5% microfluorospheres was injected into the area of the vestibular efferent nucleus, which is located lateral to the genu of the facial nerve. CGRP immunohistochemistry was processed in serial sections of the brainstem at the facial nerve genu level. Double-labeled neurons with both CGRP immunoreactivity and microfluorospheres were examined with fluorescence and confocal laser microscopy. Results: Three types of labeled neurons were observed: (1) neurons only retrogradely microfluorosphere-labeled that were mainly located in the medial vestibular nucleus, lateral vestibular nucleus, superior vestibular nucleus and parvicellular reticular nucleus on the ipsilateral side of the injection; (2) neurons that were both immunolabeled with CGRP and also retrogradedly labeled with microfluorospheres, indicating that they are CGRP cells projecting to the area of vestibular efferent nucleus, these cells were mainly distributed in the superior vestibular nucleus and dorsal vestibular nucleus, and (3) cells only immunolabeled for CGRP that were scattered extensively in the brainstem. Conclusion: The presented methodical contribution demonstrates the suitability of fluorescein-labeled microspheres for retrograde neuronal tracing. The vestibular nuclei contain numerous afferent neurons that send projections to the vestibular efferent nucleus, some of which are CGRP cells. This afferent innervation provides morphological evidence that the vestibular efferent neurons receive input from the vestibular afferent neurons including CGRP cells. These vestibular primary CGRP afferent neurons may have an influence on vestibular efferent neurons. CGRP acts as an important co-transmitter or modulator in the afferent-mediated activity of vestibular efferent neurons, which in turn affect afferents in the vestibular end organs.