Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, β1 and β1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of β1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to β1B. We show that wild-type β1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of β1B with voltage-gated Na⁺ channels Na_v1.1 or Na_v1.3 is not detectable by immunoprecipitation and β1B does not affect Na_v1.3 cell surface expression as measured by [³H]saxitoxin binding. However, β1B coexpression results in subtle alteration of Na_v1.3 currents in transfected cells, suggesting that β1B may modulate Na⁺ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of β1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that β1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.