We have previously shown that ilimaquinone (IQ), a marine sponge metabolite, causes complete vesiculation of the Golgi stacks. By reconstituting the IQ-mediated vesiculation of the Golgi apparatus in permeabilized cells, we now demonstrate that this process does not require ARF and coatomers, which are necessary for the formation of Golgi-derived COPI vesicles. We find that IQ-mediated Golgi vesiculation is inhibited by Gαs-GDP and Gαi3-GDP. Interestingly, adding βγ subunits in the absence of IQ is sufficient to vesiculate Golgi stacks. Our findings reveal that IQ-mediated Golgi vesiculation occurs through activation of heterotrimeric G proteins and that it is the free βγ, and not the activated α subunit, that triggers Golgi vesiculation.