Significance Pain development and discomfort are universal features of spider envenomation, yet severe pain arising from bites by Old World spiders is poorly understood. Molecular analyses of the venom of the King Baboon spider revealed abundant expression of the inhibitory cystine knot peptide Pm1a. Synthetic Pm1a induces pain in mice while simultaneously enhancing proexcitatory sodium currents and decreasing inhibitory potassium currents. These concomitant effects promote hyperexcitability in pain-sensing neurons that can be reversed by pharmacological inhibition of voltage-gated sodium channels. The coordinated modulation of excitatory and inhibitory ion channels involved in pain propagation may represent an economical and effective defense strategy in pain-inducing defensive venoms.