Vaccinia virus (VACV) is a valuable tool to study host-virus interactions and encodes a plethora of immunomodulatory proteins, among which three, A55, C2 and F3, have a BTB/BACK-Kelch (BBK) structure and are the focus of this study. Proteins containing BTB and Kelch domains function as adapters for the cullin 3 (Cul3)-based E3 ubiquitylation system. VACV BBKs are not essential for viral replication, but they all affect the lesion size induced by infection in an intradermal murine model. Viruses lacking any one of these BBK proteins also induce increased inflammatory cell infiltration into lesions. Deleting A55R or C2L also causes different plaque morphology and cytopathic effect in cultured cells. The mechanisms underpinning these observations were unknown, and hence were the objectives of this study. Besides, the interaction between Cul3 and VACV BBKs, the cellular substrates of A55, C2 and/or F3, and the outcome of targeting these substrates were also investigated. The effect of VACV BBKs on innate immune signalling was studied, and NF-κB was selected as a starting point because the ectromelia virus (ECTV) orthologue of A55, ECTV EVM150, inhibits NF-κB signalling. Consistent with this, A55, C2 and F3 all inhibit NF-κB signalling pathway by diminishing p65 nuclear translocation. Specifically, A55 associates with importin KPNA2 and impairs the interaction between KPNA2 and p65, hence reducing p65 importation into the nucleus. A55 interacts with Cul3 directly and with high affinity, although the ability to inhibit NF-κB is independent of binding Cul3. In contrast, C2 and F3 do not interact with Cul3. However, C2 associates with A55, and via A55, it forms a complex with Cul3. A working model was proposed in which A55, Cul3 and C2 form a complex, in which A55 binds Cul3 and C2 brings in substrates, leading to the ubiquitylation of substrates followed by protein degradation or modification. To examine this working model and to identify novel viral BBKs substrates, an unbiased tandem mass tag (TMT) mass spectrometry was carried out. Three cellular proteins, Fer, Scrib and RASA2, are degraded during infection by wild type VACV, but not by mutant VACVs lacking either C2 or A55. Thus, A55 and C2 function collaboratively. A55 and C2 degrade Scrib, and an interaction between A55 and Cul3 is required for this. Using Scrib knockout (KO) cell lines, it was found that Scrib restricts VACV spread. It was also found that VACV upregulates TGF-β signalling and Scrib influences the pathway. Besides its adaptor protein role in the A55/Cul3 complex, C2 also associates with a small GTPase named Cdc42. Cdc42 is stable during VACV infection, meaning that C2 does not degrade Cdc42 via A55/Cul3. However, Cdc42 is activated by VACV, and this activation is promoted by C2. Overall, this study shows VACV expresses the family of BBK proteins to modulate intracellular signalling and hijack host Cul3-ubiquitin system to target cellular proteins.