Systemic administration of resiniferatoxin (RTX), an ultrapotent capsaicin analogue, removes TRPV1-expressing afferent neurons and impairs thermal, but not mechanical, nociception in adult animals. In this study, we determined how loss of TRPV1-expressing sensory neurons alters the antinociceptive effect of µ opioids and µ opioid receptors in the spinal cord. The effect of morphine and (D-Ala²,N-Me-Phe⁴,Gly-ol⁵)-enkephalin (DAMGO) was measured by testing the paw mechanical withdrawal threshold in rats treated with RTX or vehicle. RTX treatment deleted TRPV1-immunoreactive dorsal root ganglion neurons and nerve terminals in the spinal dorsal horn. Also, the µ opioid receptor-immunoreactivity was markedly reduced in the superficial dorsal horn of RTX-treated rats. However, RTX treatment did not affect the dorsal horn neurons labeled with both TRPV1- and µ opioid receptor-immunoreactivity. Surprisingly, intrathecal morphine or DAMGO produced a greater increase in the withdrawal threshold in RTX- than in vehicle-treated rats. The duration of the effect of intrathecal morphine and DAMGO in RTX-treated rats was also profoundly increased. Furthermore, the antinociceptive effect of systemic morphine was significantly potentiated in RTX-treated rats. The B_MAX (but not K_D) of [³H]-DAMGO binding and DAMGO-stimulated [³⁵S]GTPS activity in the dorsal spinal cord were significantly reduced in the RTX group. This study provides novel information that loss of TRPV1 afferent neurons eliminates presynaptic mu opioid receptors present on TRPV1-expressing afferent neurons but paradoxically potentiates the analgesic effect of µ opioid agonists. Mechano-nociception, transmitted through non-TRPV1 sensory neurons, is subject to potent modulation by µ opioid agonists.