A afferent fibers have been reported to participate in the development of the central sensitization induced by inflammation and injuries. Current evidence suggests that myofascial trigger points (MTrPs) induce central sensitization in the related spinal dorsal horn, and clinical studies indicate that A fibers are associated with pain behavior. Because most of these clinical studies applied behavioral indexes, objective evidence is needed. Additionally, MTrP-related neurons in dorsal root ganglia and the spinal ventral horn have been reported to be smaller than normal, and these neurons were considered to be related to A fibers. To confirm the role of A fibers in MTrP-related central changes in the spinal dorsal horn, we studied central sensitization as well as the size of neurons associated with myofascial trigger spots (MTrSs, equivalent to MTrPs in humans) in the biceps femoris muscle of rats and provided some objective morphological evidence. Cholera toxin B subunit-conjugated horseradish peroxidase was applied to label the MTrS-related neurons, and tetrodotoxin was used to block A fibers specifically. The results showed that in the spinal dorsal horn associated with MTrS, the expression of glutamate receptor (mGluR1α/mGluR5/NMDAR1) increased, while the mean size of MTrS-related neurons was smaller than normal. After blocking A fibers, these changes reversed to some extent. Therefore, we concluded that A fibers participated in the development and maintenance of the central sensitization induced by MTrPs and were related to the mean size of neurons associated with MTrPs in the spinal dorsal horn.