[Background]: In ischemic limb, ATP and products of oxidative stress respectively stimulate purinergic P2X3 receptors and transient receptor potential cation channel V1 (TRPV1) in afferent nerves. In this study, limb ischemia was induced by 72-hour femoral artery occlusion in rats. We determined if concentration of intracellular calcium/[Ca2+]i is enhanced in muscle dorsal root ganglion (DRG) neurons of ischemic rats following stimulation of P2X3; and if removal of TRPV1 alters P2X3-increased [Ca2+]i in DRG neurons. [Methods]: Muscle DRG neurons were identified by fluorescent Dil injected previously into the hindlimb muscle. For Ca2+ measurements, fluorescence images of cells were recorded with a digital fluorescence imaging system. The ratio of fluorescence at 340 nm to that at 380 nm was obtained and F340/F380max – vehicle baseline (∆F340/F380max) for changes of [Ca2+]i. α,β-me ATP and A317491 (P2X3 antagonist) were used to activate and block P2X3. Iodoresiniferatoxin (i-RTX) was previously given to abolish TRPV1 in DRGs. [Results]: 1) A317491 decreased ∆F340/F380max in DRG neurons by α,β-me ATP (0.40±0.18/n=53; P<0.05 vs. 1.09±1.52/n=36 by α,β-me ATP alone); 2) the increase of ∆F340/F380max by α,β-me ATP was amplified in DRG neurons of ischemic group (0.87±0.75/n=107; P<0.05 vs. non-ischemia/n=92); and 3) i-RTX attenuated ∆F340/F380max increased by α,β-me ATP in ischemic group but not in non-ischemic group. [Conclusion]: The exaggeration of P2X3-evoked [Ca2+]i in muscle DRG neurons of ischemic rats is blunted by removal of TRPV1, suggesting that there is a regulatory effect of TRPV1 on the activities of P2X3 in muscle afferent nerves of ischemic limb.