Febrile seizures are a common childhood seizure disorder and a defining feature of genetic epilepsy with febrile seizures plus (GEFS+), a syndrome frequently associated with Na+ channel mutations. Here, we describe the creation of a knockin mouse heterozygous for the C121W mutation of the beta1 Na+ channel accessory subunit seen in patients with GEFS+. Heterozygous mice with increased core temperature displayed behavioral arrest and were more susceptible to thermal challenge than wild-type mice. Wild-type beta1 was most concentrated in the membrane of axon initial segments (AIS) of pyramidal neurons, while the beta1(C121W) mutant subunit was excluded from AIS membranes. In addition, AIS function, an indicator of neuronal excitability, was substantially enhanced in hippocampal pyramidal neurons of the heterozygous mouse specifically at higher temperatures. Computational modeling predicted that this enhanced excitability was caused by hyperpolarized voltage activation of AIS Na+ channels. This heat-sensitive increased neuronal excitability presumably contributed to the heightened thermal seizure susceptibility and epileptiform discharges seen in patients and mice with beta1(C121W) subunits. We therefore conclude that Na+ channel beta1 subunits modulate AIS excitability and that epilepsy can arise if this modulation is impaired.