Golgi SNARE, with a size of 28 kD (GS28), is a transmembrane protein and mainly localizes to the Golgi apparatus. It is considered a core part of the Golgi SNARE complex in the Endoplasmic Reticulum (ER)-Golgi transport and regulates the docking and fusion of transport vesicles effectively. In recent years, increasing studies have indicated that various intracellular transport events are regulated by different GS28-based SNARE complexes. Moreover, GS28 is also involved in numerous functional signaling pathways related to different diseases via interacting with other SNARE proteins and affecting protein maturation and secretion. However, the precise function of GS28 in different disease models and the regulatory network remains unclear. In this review, we mainly provide a concise overview of the function and regulation of GS28 in vesicle trafficking and diseases and summarize the signaling pathways regarding potential mechanisms. Although some critical points about the significance of GS28 in disease treatment still need further investigation, more reliable biotechnical or pharmacological strategies may be developed based on a better understanding of the diverse role of GS28 in vesicle trafficking and other biological processes.