Human immunodeficiency virus 1 (HIV‐1) expresses several accessory proteins that manipulate various host‐cell processes to achieve optimum replicative efficiency. One of them, viral protein U (Vpu), has been shown to interfere with the cellular degradation machinery through interaction with SCFβ‐TrCP complexes. To learn more about Vpu function in vivo, we used the genetically tractable fruit fly, Drosophila melanogaster. Our results show that the directed expression of Vpu, but not the non‐phosphorylated form, Vpu2/6, in fat‐body cells affects Drosophila antimicrobial responses. In flies, the Toll and Imd pathways regulate antimicrobial‐peptide gene expression. We show that Vpu specifically affects Toll pathway activation by inhibiting Cactus degradation. Given the conservation of the Toll/nuclear factor‐κB (NF‐κB) signalling pathways between flies and mammals, our results suggest a function for Vpu in the inhibition of host NF‐κB‐mediated innate immune defences and provide a powerful genetic approach for studying Vpu inhibition of NF‐κB signalling in vivo.