Ets proteins constitute a family of conserved sequence-specific DNA-binding proteins and function as transcription factors. ETS1 plays important roles in differentiation, lymphoid cell development, invasiveness and angiogenesis. Such diverse roles of ETS1 are likely to be dependent on its associated proteins. A yeast two-hybrid screen was conducted and here we describe a novel ETS1 interacting protein designated as ETS1-associated protein II (EAPII). EAPII protein interacts with ETS1 and other Ets proteins (ETS2 and FLI1) both in vitro and in vivo. Indirect immunofluorescence demonstrated that EAPII is predominately localized to the nucleus of mammalian cells. EAPII negatively modulates ETS1 transcriptional activity and attenuates synergistic transactivation by ETS1 and AP-1. Significantly, re-expression of EAPII inhibits the migration of epithelial cancer cells, but does not affect cell viability. Therefore, EAPII is a novel ETS1 modulator that regulates specific aspects of the ETS1 functions.