Expression of Q205L Gα_o(Gα_o*), an alpha subunit of heterotrimeric guanine nucleotide–binding proteins (G proteins) that lacks guanosine triphosphatase (GTPase) activity in NIH-3T3 cells, results in transformation. Expression of Gα_o* in NIH-3T3 cells activated signal transducer and activator of transcription 3 (Stat3) but not mitogen-activated protein (MAP) kinases 1 or 2. Coexpression of dominant negative Stat3 inhibited Gα_o*-induced transformation of NIH-3T3 cells and activation of endogenous Stat3. Furthermore, Gα_o* expression increased activity of the tyrosine kinase c-Src, and the Gα_o*-induced activation of Stat3 was blocked by expression of Csk (carboxyl-terminal Src kinase), which inactivates c-Src. The results indicate that Stat3 can function as a downstream effector for Gα_o* and mediate its biological effects.