In our previous study, fluoride ([AlF4]−) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of Gi protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, Gi2 protein was detected on the microsome membrane by western blotting for heterotrimeric Gi proteins. These results indicate that ER-Gi2 protein modulated Sar1 translocation onto the ER, suggesting that ER-resident Gi2 protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER. J. Cell. Biochem. 112: 2250–2256, 2011. © 2011 Wiley-Liss, Inc.