Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in neurons. The human genome includes ten human VGSC α-subunit genes, SCN(X)A, encoding Na_v1.1–1.9 plus Na_x. To understand the unique role that each VGSC plays in normal and pathophysiological function in neural networks, compounds with high affinity and selectivity for specific VGSC subtypes are required. Toward that goal, a structural analog of the VGSC pore blocker tetrodotoxin, 4,9-anhydrotetrodotoxin (4,9-ah-TTX), has been reported to be more selective in blocking Na⁺ current mediated by Na_v1.6 than other TTX-sensitive VGSCs, including Na_v1.2, Na_v1.3, Na_v1.4, and Na_v1.7. While SCN1A, encoding Na_v1.1, has been implicated in several neurological diseases, the effects of 4,9-ah-TTX on Na_v1.1-mediated Na⁺ current have not been tested. Here, we compared the binding of 4,9-ah-TTX for human and mouse brain preparations, and the effects of 4,9-ah-TTX on human Na_v1.1-, Na_v1.3- and Na_v1.6-mediated Na⁺ currents using the whole-cell patch clamp technique in heterologous cells. We show that, while 4,9-ah-TTX administration results in significant blockade of Na_v1.6-mediated Na⁺ current in the nanomolar range, it also has significant effects on Na_v1.1-mediated Na⁺ current. Thus, 4,9-ah-TTX is not a useful tool in identifying Na_v1.6-specific effects in human brain networks.