Apoptosis, programmed cell death, is a major cellular activity and is highly involved in metazoan development, inflammatory responses, tumor growth control, and many other processes. There are strong connections between apoptosis and intracellular metabolism. Metabolites such as free fatty acids (FFAs) have distinct roles in regulating cellular apoptosis: saturated FFAs (e.g. palmitate) induce apoptosis in cardiac cells, pancreatic beta cells, and hepatocytes, while unsaturated FFAs (e.g. oleate) protect cells from apoptosis induced by saturated FFAs by restoring the cardiolipin levels and diverting the saturated FFAs to triglyceride accumulation. Palmitate-induced apoptosis is accompanied by the accumulation of ceramide, altered protein level of Bcl-2, and activity level of NF-kB. However, the mechanism by which palmitate signals cellular apoptosis remains unclear. In the present study we identified that the double-stranded RNA-dependent protein kinase (PKR) is involved, as well as how it is involved, in mediating palmitate-induced apoptosis of human hepatocellular carcinoma cell (HepG2). PKR acts as an apoptotic factor in many different types of cells, but more recently, an anti-apoptotic role has been attributed to PKR in tumor cells including human breast cancer cells and HCV-related hepatocellular carcinoma. Similarly, we confirmed that PKR has an anti-apoptotic role in HepG2 cells, and identified for the first time that palmitate down-regulates the activity of PKR, which we observed together with a decrease in the Bcl-2 protein level. Using pharmaceutical inhibitors of PKR activity and siRNA of the PKR gene, we established that PKR is required to maintain the protein level of Bcl-2 in HepG2 cells. In addition, we confirmed that the transcription factor, NF-kB, which can be activated by PKR, is involved in mediating the effect of PKR on Bcl-2 level. The phosphorylation of Bcl-2 at the anti-apoptotic site, Ser70, sustains the anti-apoptotic role of Bcl-2, while its pro-apoptotic site, Ser87, attenuates the anti-apoptotic role. We found that PKR increased the phosphorylation of Bcl-2 at Ser70 and repressed that of Ser87, showing a dual effect on the phosphorylation of Bcl-2. Since Bcl-2 has consensus motifs for both PP2A and MAPKs, the effect we observed could result from the simultaneous activation by PKR of protein phosphatase, PP2A, and protein kinases, MAPKs. However, through co-immunoprecipitation analysis we identified for the first time that PKR interacts directly with Bcl-2, suggesting that PKR, a Ser/Thr kinase, also plays a direct role in inducing the phosphorylation of Bcl-2. By over-expressing the site-directed mutants of Bcl-2 protein, we identified the phosphorylation sites of Bcl-2 that are essential for the direct interaction between PKR and Bcl-2. Finally, we constructed a network model of metabolic and signal transduction pathways incorporating the novel connections between PKR and Bcl-2 that reveals how intracellular metabolites, signaling molecules, and exogenous palmitate regulate human hepatocellular carcinoma apoptosis.