Viral infections produce double-stranded (ds)RNAs or RNAs containing duplex regions that activate the protein kinase R (PKR), an interferon-induced protein. Activated PKR phosphorylates the eukaryotic translation initiation factor 2α (eIF2α), leading to translation inhibition, affecting viral replication of RNA viruses, and the modulation of the induction of innate immune responses. The regulation of PKR functions is not well known, so a better understanding is essential. Here, through co-immunoprecipitation analyses, we have identified for the first time an interaction between IFN alpha inducible protein 27 (IFI27) and PKR-activating protein (PACT or PRKRA) and with PKR, and these interactions are mediated by the presence of dsRNAs or RNAs containing duplex regions. Furthermore, we have shown that the interaction of IFI27 with PKR is PACT-dependent. The interaction of IFI27 with PKR and PACT, led to stronger activation of PKR and, therefore, a decrease in protein translation, both after treatment with the dsRNA analogue poly(I:C), and with different RNA viruses such as Severe Acute Respiratory virus 2 (SARS-CoV-2) and Vesicular Stomatitis virus (VSV). We also show that the function of IFI27 to activate PKR is dependent on its interaction with PACT. Moreover, we show that IFI27 expression increases the formation of stress granules, correlating with the increased PKR activation mediated by IFI27, as it has been shown that the translational arrest induced by activated PKR leads to the formation of stress granules. IFI27 positively modulates PKR activation in a PACT-dependent manner. This effect is broad, as it is shown for infections with SARS-CoV-2 and VSV, and with poly(I:C) treatment. Further understanding of the regulation of PKR activity will allow us to develop new antiviral drugs to modulate this signalling axis, which is crucial in RNA virus infections.