Syntaxin 1C (STX1C), produced by alternative splicing of the stx1A gene, is a soluble syntaxin lacking a SNARE domain and a transmembrane domain. It is unclear how soluble syntaxin can control intracellular membrane trafficking. We found that STX1C affected microtubule (MT) dynamics through its tubulin-binding domain (TBD) and regulated recycling of intracellular vesicles carrying glucose transporter-1 (GLUT1). We demonstrated that the amino acid sequence VRSK of the TBD was important for the interaction between STX1C and tubulin and that wild-type STX1C (STX1C-WT), but not the TBD mutant, reduced the V(max) of glucose transport and GLUT1 translocation to the plasma membrane in FRSK cells. Moreover, by time-lapse analysis, we revealed that STX1C-WT suppressed MT stability and vesicle-transport motility in cells expressing GFP-α-tubulin, whereas TBD mutants had no effect. We also identified that GLUT1 was recycled in the 45 minutes after endocytosis and that GLUT1 vesicles moved along with MTs. Finally, we showed, by a recycling assay and FCM analysis, that STX1C-WT delayed the recycling phase of GLUT1 to PM, without affecting the endocytotic process of GLUT1. These data indicate that STX1C delays the GLUT1 recycling phase by suppressing MT stability and vesicle-transport motility through its TBD, providing the first insight into how soluble syntaxin controls membrane trafficking.