TRIM25, an E3 ligase, is an important regulator to modulate the functions of retinoic acid inducible gene-I (RIG-I) and other factors in innate immunity. Herein the structural interaction between the 2CARD domain of RIG-I and the PRYSPRY domain of TRIM25 was investigated by nuclear magnetic resonance, X-ray crystallography, computer-assisted modeling, and cell-based assays to elucidate the complex structure of PRYSPRY/2CARD. The interacting model indicated that docking of 2CARD onto PRYSPRY brought two RIG-I molecules into a close proximity to form a dimer. The attachment of a short ubiquitin chain covalently by the TRIM25's E3 ligase activity was favorable for tethering a neighboring RIG-I dimer to form the tetrameric RIG-I by non-covalent interactions. The data supported the notion that the TRIM25/RIG-I interaction was important to activate the RIG-I pathway to suppress the replication of RNA viruses, such as vesicular stomatitis virus. This work provides a structural rationale to delineate the underlying mechanism of TRIM25 regulation of RIG-I.