Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-associated proteins are a class of transmembrane proteins involved in intracellular trafficking pathways. However, the functions of many SNARE domain-containing proteins remain unclear. We have previously identified a SNARE-associated gene in alfalfa (Medicago sativa ) KILLING ME SLOWLY1 (MsKMS1 ), which is involved in various abiotic stresses. In this study, we investigated the function of MsKMS1 in the seed germination of transgenic tobacco (Nicotiana tabacum ). Phylogenetic analysis showed that MsKMS1 was homologous to the SNARE-associated or MAPR component-related proteins of other plants. Germination assays revealed that MsKMS1 negatively regulated seed germination under normal, D-mannitol and abscisic acid-induced stress conditions, yet MsKMS1 -overexpression could confer enhanced heat tolerance in transgenic tobacco. The suppressive effect on germination in MsKMS1 -overexpression lines was associated with higher abscisic acid and salicylic acid contents in seeds. This was accompanied by the upregulation of abscisic acid biosynthetic genes (ZEP and NCED ) and the downregulation of gibberellin biosynthetic genes (GA20ox2 and GA20ox3 ). Taken together, these results suggested that MsKMS1 negatively regulated seed germination by increasing abscisic acid and salicylic acid contents through the expression of genes related to abscisic acid and gibberellin biosynthesis. In addition, MsKMS1 could improve heat tolerance during the germination of transgenic tobacco seeds.