Adaptive evolution in response to one selective challenge may disrupt other important aspects of performance. Such evolutionary trade-offs are predicted to arise in the process of local adaptation, but it is unclear if these phenotypic compromises result from the antagonistic effects of simple amino acid substitutions. We tested for trade-offs associated with beneficial mutations that confer tetrodotoxin (TTX) resistance in the voltage-gated sodium channel (NaV1.4) in skeletal muscle of the common garter snake (Thamnophis sirtalis). Separate lineages in California and the Pacific Northwest independently evolved TTX-resistant changes to the pore of NaV1.4 as a result of arms race coevolution with toxic prey, newts of the genus Taricha. Snakes from the California lineage that were homozygous for an allele known to confer large increases in toxin resistance (NaV1.4LVNV) had significantly reduced crawl speed compared to individuals with the ancestral TTX-sensitive channel. Heterologous expression of native snake NaV1.4 proteins demonstrated that the same NaV1.4LVNV allele confers a dramatic increase in TTX resistance and a correlated decrease in overall channel excitability. Our results suggest the same mutations that accumulate during arms race coevolution and beneficially interfere with toxin-binding also cause changes in electrophysiological function of the channel that may affect organismal performance. This trade-off was only evident in the predator lineage where coevolution has led to the most extreme resistance phenotype, determined by four critical amino acid substitutions. If these biophysical changes also translate to a fitness cost—for example, through the inability of T. sirtalis to quickly escape predators—then pleiotropy at this single locus could contribute to observed variation in levels of TTX resistance across the mosaic landscape of coevolution.