SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex formation is necessary for intracellular membrane fusion and thus has a key role in processes such as secretion. However, little is known about the regulatory factors that bind to Qa-SNAREs, which are also known as syntaxins (SYPs) in plants. Here, we characterized Arabidopsis (Arabidopsis thaliana) Tomosyn protein (AtTMS) and demonstrated that it is a conserved regulator of SYPs in plants. AtTMS binds strongly via its R-SNARE motif-containing C terminus to the Qa domain of PM-resident, pollen-expressed SYP1s (SYP111, SYP124, SYP125, SYP131, and SYP132), which were narrowed down from 12 SYPs. AtTMS is highly expressed in pollen from the bicellular stage onwards, and overexpression of AtTMS under the control of the UBIQUITIN10, MSP1, or LAT52 promoter all resulted in defective pollen after the microspore stage in which secretion was inhibited, leading to the failure of intine deposition and cell plate formation during pollen mitosis I. In tobacco (Nicotiana benthamiana) leaf epidermal cells, overexpression of AtTMS inhibited the secretion of secreted GFP. The defects were rescued by mCherry-tagged SYP124, SYP125, SYP131, or SYP132. In vivo, SYP132 partially rescued the pMSP1:AtTMS phenotype. In addition, AtTMS, lacking a transmembrane domain, was recruited to the plasma membrane by SYP124, SYP125, SYP131, and SYP132 and competed with Vesicle-Associated Membrane Protein721/722 for binding to, for example, SYP132. Together, our results demonstrated that AtTMS might serve as a negative regulator of secretion, whereby active secretion might be fine-tuned during pollen development.