The syntaxins are proteins associated with various intracellular membrane compartments. They are major participants in a large variety of physiological processes where membrane fusion occurs, including exocytosis. We have identified a novel syntaxin isoform generated by alternative splicing of the human STX1B gene. In contrast with the canonical syntaxins, this isoform (STX1B-DeltaTMD) lacked the classical C-terminal transmembrane domain and localized to the nucleus of various tumoral and non-tumoral cell types including human brain cortical neurons in vivo. The reversible blockade of STX1B-DeltaTMD nuclear import demonstrated that nuclear import occurred via a Ran-dependent pathway. A specific and glycine-rich C-terminus of 15 amino acids served as an unconventional nuclear localization signal. STX1B-DeltaTMD colocalized with Lamin A/C and NuMA (NUclear Mitotic Apparatus protein) in interphasic nuclei, and with NuMA and gamma-tubulin in the pericentrosomal region of the mitotic spindle in dividing cells. In a series of 37 human primary brain tumors, the ratio of STX1B-DeltaTMD to Lamin A/C transcripts was a significant prognostic marker of survival, independent of tumor staging. The characterization of STX1B-DeltaTMD as the first nucleoplasmic syntaxin with no transmembrane domain, illustrates the importance of alternative splicing in the emergence of unsuspected properties of the syntaxins in human cells, in both physiological and pathological conditions.