Spider venom is rich in peptide toxins that could be used to explore the structure and function of voltage-gated sodium channels (NaVs). This study has characterized a 44-amino acid peptide toxin, δ-hexatoxin-MrIX (δ-HXTX-MrIX), from the venom of the spider Macrothele raveni. δ-hexatoxin-MrIX potently inhibited the fast inactivation of NaVs in mouse cerebellar granule cells (CGCs) with an EC50 of 35.3±5.9nM. The toxin shifted both the steady-state activation and the steady-state inactivation curves of CGC NaVs to the hyperpolarized direction. δ-hexatoxin-MrIX also acted on NaV1.3 and NaV1.4 channels heterologously expressed in HEK293T cells, as well as on NaVs in acutely isolated cockroach DUM neurons. However, the NaV1.5, NaV1.7 and NaV1.8 channels were resistant to δ-hexatoxin-MrIX. The toxin inhibited the fast inactivation of NaV1.3 and NaV1.4 with high affinity (EC50 values of 82.0±3.0nM and 24.0±4.7nM, respectively), but the saturating dose of toxin showed distinct efficacy on these two types of channels. δ-hexatoxin-MrIX is a peptide toxin acting on CGC NaVs and could be used as a pharmacological tool to explore the role of NaVs in granule cell maturation during cerebellum development.