Inositol requiring enzyme 1 (IRE1α) is a sensor that responds to endoplasmic reticulum stress with self‐phosphorylation, oligomerization and RNase activity. As a result, IRE1α activates the critical transcription factor XBP1, while reducing the expression of select other transcripts, to cope with the level of ER stress. In response to stress IRE1α molecules also cluster transiently, with similar time frame to the enzymatic activity.The response of IRE1α is inhibited by mutations in the kinase and RNase active sites of IRE1α‐, but not by most mutations with no direct catalytic role. Here we describe amino acid substitutions in the segment connecting the kinase and RNase domains which affect IRE1α clustering, its RNase activities or both. The more detrimental substitutions involve Pro mutations and they do not support RNase activity, fail to auto‐phosphorylate in response to stress and do not oligomerize. The substitution L827P is dominant‐negative in vivo: it dimerizes with full‐length WT IRE1α and consequently inhibits its activation in lymphoma and multiple myeloma cells. Expression of the IRE1α L827P mutant also increases the sensitivity of lymphoma cells to ER stress and inhibits their growth. Less drastic substitutions, such as L827F have intact RNase and kinase activities but fails to oligomerize and displays altered self‐phosphorylation, in particular on Ser729. Higher sensitivity to partial proteolysis indicates directly that the conformations of L827P, L827F and WT IRE1α are distinct.Furthermore, our data show that clustering of IRE1α depends on sensing ER stress but not on its RNase activity. There are conditions that initiate IRE1α RNase activity without inducing clustering, and conversely ‐ RNase‐impaired molecules can cluster. We hypothesize that depending on which residues are phosphorylated, IRE1α assumes different conformations, only some of which are permissive for clustering. Therefore, this work shows that the kinase‐RNase connector is important for attaining the stress‐responsive, clustering‐capable conformation of IRE1α and that co‐expression of variant IRE1α sensors profoundly inhibits the normal signaling.Support or Funding InformationT32 HL 7954‐19 (R.D.)