AbstractTwo types of syntaxin 1 isoforms, HPC‐1/syntaxin 1A (STX1A) and syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A−/− mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B−/− mice using targeted gene disruption and investigated their phenotypes. STX1B−/− mice were born alive, but died before postnatal day 14, unlike STX1A−/− mice. Morphologically, brain development in STX1B−/− mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B−/− neurons was lower than that of WT or STX1A−/− neurons after 9 days. Interestingly, STX1B−/− neurons survived on WT or STX1A−/− glial feeder layers as well as WT neurons. However, STX1B−/− glial feeder layers were less effective at promoting survival of STX1B−/− neurons. Conditioned medium from WT or STX1A−/− glial cells had a similar effect on survival, but that from STX1B−/− did not promote survival. Furthermore, brain‐derived neurotrophic factor (BDNF) or neurotrophin‐3 supported survival of STX1B−/− neurons. BDNF localization in STX1B−/− glial cells was disrupted, and BDNF secretion from STX1B−/− glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia. image Syntaxin 1A (STX1A) and syntaxin 1B (STX1B) are thought to have similar functions as SNARE proteins. However, we found that STX1A and STX1B play distinct roles in neuronal survival using STX1A−/− mice and STX1B−/− mice. STX1B was important for neuronal survival, possibly by regulating the secretion of neurotrophic factors, such as BDNF, from glial cells.