Extracellular signals perceived by G protein-coupled receptors are transmitted via G proteins and subsequent intracellular signaling cascades result in a plethora of physiological responses. The natural product cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the Gq subfamily of G proteins, the X-ray crystallography structure of YM-254890 bound to a chimeric Gq/i protein demonstrated that YM-254890 binds to a hydrophobic cavity and stabilizes an inactive guanosine diphosphate (GDP)-bound form of the Gq protein. However, no synthetic route has been reported previously for these complex natural products and they are not easily isolated from natural sources. Here we report the first total synthesis of YM-254890 and FR900359, as well as of two known analogues, YM-385780 and YM-385781. The versatility of the synthetic approach also enabled the design and synthesis of ten analogues, which provided the first structure–activity relationship study for this class of compounds. Pharmacological characterization of all the compounds at Gq-, Gi- and Gs-mediated signalling provided succinct information on the structural requirements for inhibition, and demonstrated that both YM-254890 and FR900359 are highly potent inhibitors of Gq signalling, with FR900359 being the most potent. These natural products and their analogues represent unique tools for explorative studies of G protein inhibition.