Syntaxins are a conserved family of SNARE proteins and contain C-terminal transmembrane anchors required for their membrane fusion activity. Here we show that syntaxin 3 (Stx3) unexpectedly also functions as a nuclear regulator of gene expression. We found that alternative splicing creates a soluble isoform, we termed Stx3S, lacking the transmembrane anchor. Soluble Stx3S binds to the nuclear import factor RAN-binding protein 5 (RanBP5), targets to the nucleus and interacts physically and functionally with several transcription factors, including ETS variant 4 (ETV4) and activating transcription factor 2 (ATF2). Stx3S is differentially expressed in normal human tissues, during epithelial cell polarization, and in breast cancer versus normal breast tissue. Inhibition of endogenous Stx3S expression alters the expression of cancer-associated genes and promotes cell proliferation. Similar nuclear-targeted, soluble forms of other syntaxins were identified suggesting that nuclear signaling is a conserved, novel function common among these membrane trafficking proteins.