Phospholipase Cε (PLCε) cleaves phosphatidylinositol lipids to increase intracellular Ca 2+ and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLCε is activated via direct binding of small GTPases at the cytoplasmic leaflets of cellular membranes. In the cardiovascular system, the RhoA GTPase regulates PLCε to initiate a cardioprotective pathway, but the underlying molecular mechanism is not known. We present here the cryo-electron microscopy (cryo-EM) reconstruction of RhoA bound to PLCε. The G protein binds a unique insertion in the PLCε EF hands. Deletion of or mutations to this PLCε insertion decrease RhoA-dependent activation without impacting regulation by other G proteins. Together, our data support a model wherein RhoA binding to PLCε allosterically activates the lipase and increases its interactions with the membrane, resulting in maximum activity and cardiomyocyte survival.