Initially identified to be activated upon virus infection, the double-stranded RNA-dependent protein kinase (PKR) is best known for triggering cell defense responses by phosphorylating eIF-2α, thus suppressing RNA translation. We as well as others showed that the phosphorylation of PKR is down-regulated by insulin. In the present study, we further uncovered a novel function of PKR in regulating the IRS proteins. We found that PKR up-regulates the inhibitory phosphorylation of IRS1 at Ser312, which suppresses the tyrosine phosphorylation of IRS1. This effect of PKR on the phosphorylation of IRS1 is mediated by two other protein kinases, JNK and IKK. In contrast, PKR regulates IRS2, another major IRS family protein in the liver, at the transcriptional rather than the posttranslational level, and this effect is mediated by the transcription factor, FoxO1, which has been previously shown to be regulated by insulin and plays a significant role in glucose homeostasis and energy metabolism. In summary, we found for the first time that initially known as a virus infection response gene, PKR regulates the upstream central transmitters of insulin signaling, IRS1 and IRS2, through different mechanisms.