Mechanical and metabolic stimuli within contracting skeletal muscles evoke reflex autonomic and cardiovascular adjustments. In cats and rats, gadolinium has been used to investigate the role played by the mechanical component of this reflex, termed the exercise pressor reflex. Gadolinium, however, has poor selectivity for mechano-gated channels and exerts multiple off-target effects. We tested the hypothesis that GsMTX4, a more selective mechano-gated channel inhibitor than gadolinium and a particularly potent inhibitor of mechano-gated Piezo channels, reduced the exercise pressor reflex in decerebrate rats. Injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 5, GsMTx4: 12 ± 5 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to tendon stretch, a purely mechanical stimulus, but had no effect on the pressor responses to intra-arterial injection of α,β-methylene ATP or lactic acid. Moreover, injection of 10 μg of GsMTx4 into the arterial supply of the hindlimb reduced the peak pressor (control: 24 ± 2, GsMTx4: 14 ± 3 mmHg, P < 0.01), cardioaccelerator and renal sympathetic nerve responses to electrically-induced intermittent hindlimb muscle contractions. By contrast, injection of 10 μg of GsMTx4 into the jugular vein had no effect on the pressor, cardioaccelerator, or renal sympathetic nerve responses to contraction. Quantitative RT-PCR and western blot analyses indicated that both Piezo1 and Piezo2 channel isoforms were natively expressed in rat dorsal root ganglia tissue. We conclude that GsMTx4 reduced the exercise pressor reflex in decerebrate rats and that the reduction was attributable, at least in part, to its effect on mechano-gated Piezo channels.