Gβγ subunits play key roles in modulation of canonical effectors in G protein-coupled receptor (GPCR)-dependent signalling at the cell surface. However, a number of recent studies of Gβγ function have revealed that they regulate a large number of molecules at distinct subcellular sites. These novel, non-canonical Gβγ roles have reshaped our understanding of how important Gβγ signalling is compared to our original notion of Gβγ subunits as simple negative regulators of Gα subunits. Gβγ dimers have now been identified as regulators of transcription, anterograde and retrograde trafficking and modulators of second messenger molecule generation in intracellular organelles. Here, we review some recent advances in our understanding of these novel non-canonical roles of Gβγ.