Double-stranded RNA-dependent protein kinase (PKR), a ubiquitously expressed serine/threonine kinase, has been implicated in the regulation or modulation of cell growth through multiple signaling pathways, but how PKR regulates tumor necrosis factor (TNF)-induced signaling pathways is poorly understood. In the present study, we used fibroblasts derived from PKR gene-deleted mice to investigate the role of PKR in TNF-induced activation of nuclear factor-κB (NF-κB), mitogen-activated protein kinases (MAPKs) and growth modulation. We found that in wild-type mouse embryonic fibroblast (MEF), TNF induced NF-κB activation as measured by DNA binding but deletion of PKR abolished this activation. This inhibition was associated with suppression of inhibitory subunit of NF-κB (IκB)α kinase (IKK) activation, IκBα phosphorylation and degradation, p65 phosphorylation and nuclear translocation, and NF-κB-dependent reporter gene transcription. TNF-induced Akt activation needed for IKK activation was also abolished by deletion of PKR. NF-κB activation was diminished in PKR-deleted cells transfected with TNF receptor (TNFR) 1, TNFR-associated death domain and TRAF2 plasmids; NF-κB activated by NF-κB-inducing kinase, IKK or p65, however, was minimally affected. Among the MAPKs, it was interesting that whereas TNF-induced c-Jun N-terminal kinase (JNK) activation was abolished, activation of p44/p42 MAPK and p38 MAPK was potentiated in PKR-deleted cells. TNF induced the expression of NF-κB-regulated gene products cyclin D1, c-Myc, matrix metalloproteinase-9, survivin, X-linked inhibitor-of-apoptosis protein (IAP), IAP1, Bcl-xL, A1/Bfl-1 and Fas-associated death domain protein-like IL-1β-converting enzyme-inhibitory protein in wild-type MEF but not in PKR−/− cells. Similarly, TNF induced the proliferation of wild-type cells, but this proliferation was completely suppressed in PKR-deleted cells. Overall, our results indicate that PKR differentially regulates TNF signaling; IKK, Akt and JNK were positively regulated, whereas p44/p42 MAPK and p38 MAPK were negatively regulated.