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Na_V1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of <i>Tityus fasciolatus</i>, has been reported to selectively activate Na_V1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for Na_V1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of Na_V1.3 activation in the hyperpolarizing direction by −11.3 mV, with no activity at Na_V1.1, Na_V1.2, and Na_V1.4-Na_V1.8. Additional activity was found at Na_V1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by −6.3 mV. In an attempt to convert Tf2 into an Na_V1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at Na_V1.3, although it still caused a small −5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the Na_V1.1/1.3 inhibitor ICA-121431 nor in Na_V1.9^-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at Na_V1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.