Retinoic-acid inducible gene I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the immune response against RNA viruses. Upon ligand binding, RIG-I undergoes a conformational change for homodimerization, and this RNA ligand-induced dimerization is a prerequisite of its induction of downstream innate immune signalling. The repressor domain (RD) of RIG-I governs RIG-I conformational change and RIG-I dimerization; however, how RIG-I dimerization is regulated beyond these processes is unclear. Here we report that reversible acetylation modifications of lysine residues in the RD controls RIG-I activation. Acetyl-mimetic mutants of RIG-I failed to signal downstream to activate IRF-3 in response to acute SenV infection, while HCV RNA recognition and binding activities of RIG-I were intact. Acetyl-mimetic mutants of RIG-I could not form virus-induced homodimers, suggesting that acetyl-lysine residues in the resting cells prevent RIG-I from homodimerization. During acute infection, deacetylation of RIG-I RD occurs and promotes RIG-I dimerization upon ligand binding. Our preliminary data indicated that the cytoplasmic deacetylase, Histone deacetylase 6 (HDAC6), may be involved in deacetylation of RIG-I during acute infection. HDAC6 knock-down cells were impaired in RIG-I-dependent IFN-beta promoter activity during SenV infection, and overexpression of HDAC6 accelerated the kinetics of RIG-I activation in response to infection. Our results define deacetylation as a key step in controlling RIG-I activation required for innate immunity.