In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an important role in growth, multiplication, differentiation and participates in inflammation, tumorigenesis, metabolic disorders and immune response. STAT3 is a protein that shuttles between the nucleus and cytoplasm. Compared to the STAT3 in cell nucleus, we did not know the function of STAT3 in cytoplasm for a long time. Some recent studies have shown that cytoplasmic STAT3 regulates autophagy through the interaction with the double-stranded RNA-activated protein kinase (PKR), which plays an important role in cellular antiviral response. Fish is a good target for developmental and comparative immunology. In the present study, we found that the expression of grass carp (Ctenopharyngodon idella) STAT3 (CiSTAT3) was ubiquitous and significantly up-regulated under the stimulation of poly I:C. To explore the potential function of fish cytoplasmic STAT3 in the antiviral signaling pathways, in this paper we analyzed the relationship between cytoplasmic CiSTAT3 and CiPKR. We demonstrated that the CiSTAT3 can combine with CiPKR in vivo and in vitro. The SH2 domain of CiSTAT3 and the C-terminus of CiPKR play an important role in this process. Moreover, the dimer of CiSTAT3 and CiPKR was formed under normal circumstances, however, it was dissociated under the induction of poly I:C. So, we guessed the binding of CiSTAT3 and CiPKR may regulate cell viability. It has also been shown that overexpression of CiSTAT3 in CIK cells can significantly reduce the level of p-eIF2α. On the contrary, the siRNA-mediated knockdown of CiSTAT3 and Stattic induction in CIK cells can up-regulate the p-eIF2α level. To further understand the relationship between CiSTAT3 and p-eIF2α level, we carried out the CiPKR-knockdown experiment. The result indicated that CiSTAT3 regulated the level of p-eIF2α through binding to CiPKR. In addition, overexpression of CiSTAT3 in CIK cells was able to improve the cell viability. These results above unraveled the molecular mechanism of fish cytoplasmic STAT3 regulating the eIF2α phosphorylation and cell viability. Therefore, the function of fish cytoplasmic STAT3 is similar to those of mammals.