This study shows that stimulation of a range of innate and adaptive immune receptors results in the accumulation of ubiquitylated components of the nuclear factor-κB (NF-κB) signalling cascade on the cytoplasmic leaflet of the endoplasmic reticulum (ER) membrane. ER membrane fractions from stimulated cells could activate inhibitor of NF-κB kinase (IKK) in a cell-free system, which indicates that the ER membrane anchors a signalosome that is sufficient to propagate NF-κB signalling. The ER-resident protein metadherin was shown to associate with ubiquitylated NF-κB signalling components, and knockdown of metadherin in both B and T cells inhibited the accumulation of ubiquitylated signalling components on the ER and selectively decreased NF-κB activation downstream of various immune receptors. The results support a role for the ER in outside-in signalling.