Arabidopsis thaliana hit1-1 is a heat-intolerant mutant. The HIT1 gene encodes a protein that is homologous to yeast Vps53p, which is a subunit of the Golgi-associated retrograde protein (GARP) complex that is involved in retrograde membrane trafficking to the Golgi. To investigate the correlation between the cellular role of HIT1 and its protective function in heat tolerance in plants, it was verified that HIT1 was co-localized with AtVPS52 and AtVPS54, the other putative subunits of GARP, in the Golgi and post-Golgi compartments in Arabidopsis protoplasts. A bimolecular fluorescence complementation assay showed that HIT1 interacted with AtVPS52 and AtVPS54, which indicated their assembly into a protein complex in vivo. Under heat stress conditions, the plasma membrane of hit1-1 was less stable than that of the wild type, as determined by an electrolyte leakage assay, and enhanced leakage occurred before peroxidation injury to the membrane. In addition, the ability of hit1-1 to survive heat stress was not influenced by exposure to light, which suggested that the heat intolerance of hit-1 was a direct outcome of reduced membrane thermostability rather than heat-induced oxidative stress. Furthermore, hit1-1 was sensitive to the duration (sustained high temperature stress at 37°C for 3d) but not the intensity (heat shock at 44°C for 30min) of exposure to heat. Collectively, these results imply that HIT1 functions in the membrane trafficking that is involved in the thermal adaptation of the plasma membrane for tolerance to long-term heat stress in plants.