ABSTRACT TRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25’s E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA-binding have been described, the precise RNA binding site has not been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. Mutational analysis shows that RNA binding affects ubiquitination of RIG-I in mammalian cells. In addition, we present a simple model system for RNA-induced liquid-liquid phase separation of TRIM25 in vitro , resembling previously observed cellular RNA granules, that facilitates the recruitment of RIG-I.