The Sec1/Munc18 (SM) protein family plays an essential role in vesicle fusion processes of eukaryotic cells. The SM proteins are suggested to function in the regulation of SNARE-mediated vesicle fusion, primarily by binding to the SNARE protein syntaxin (Syx). Munc18a, the SM protein involved in synaptic exocytosis, binds to the closed conformation as well as the N-peptide of Syx1a, while some other SM proteins are supposed to bind only the N-peptide. Munc18c, a close homolog of Munc18a, is a ubiquitously expressed vertebrate protein that has a preference for Syx4. It is mainly studied in regulated exocytosis of GLUT4 vesicles in response to insulin. The crystal structure of Munc18c is solved in association with the N-peptide of Syx4, but its interaction with rest of the Syx4 protein is not clear. Also, the influence of Munc18c on SNARE complex formation is debated. We now investigate the Munc18c-Syx4 binding mode and its influence on SNARE assembly kinetics using biochemical and biophysical methods. Our analyses indicate that Munc18c also interacts with the “closed conformation” of Syx4. Moreover, the presence of Munc18c slows down the SNARE assembly reaction. Vertebrates have three Munc18 isoforms involved in exocytosis in different tissues that interact with four secretory syntaxin isoforms, but their interaction patterns are not mapped clearly. Since SM proteins are suggested to give specificity to the SNARE interaction, the preference of the secretory SM proteins for the syntaxin isoforms was also studied.