The E3L protein of vaccinia virus (VV) is well known for its capacity to evade cellular innate antiviral immunity related to interferon (IFN), for example PKR and RNaseL mediated antiviral activities. However, due to the limited range of cells that support VV E3L deletion mutant replication, the full capacity of E3L inhibiting the innate immune response induced by IFNs remains to be examined. In this report, the inhibition activity of VV E3L against a wide spectrum of human IFNs, including type I IFNs (12 IFN-alpha subtypes, IFN-beta, and IFN-omega), and type II IFN (gamma), was comparatively examined using the Copenhagen strain E3L deletion mutant and its revertant control virus in a human hepatoma cell line, Huh7. Deletion of the E3L open reading frame rendered the mutant VV sensitive to all types of IFNs, while the revertant VV was strongly resistant to these treatments. Furthermore, we show that the inhibition of VV E3L deletion mutant by IFN occurs at the stage of intermediate gene translation, while the expression of early genes and transcription of intermediate genes are largely unaffected. Using specific siRNAs to suppress the classical IFN-induced antiviral pathways, we found that PKR is the key factor modulated by E3L, while the RNaseL and MxA pathways play limited roles in this Huh7 cell system. Thus, our data demonstrates that VV E3L can mediate strong inhibition activity against all human type I and type II IFNs, mainly through modulation of the PKR pathway in Huh7 cells.