The 3′UTR in the KDM6B mRNA can control the cellular activity of the produced KDM6B/JMJD3 protein, but the mechanism is unclear. To examine whether 3′UTR-dependent JMJD3 protein complexes are responsible for the observed 3′UTR-dependent protein activity, KDM6B cDNA expression constructs with or without the KDM6B 3′UTR were transfected into HeLa cells grown in SILAC media, followed by co-IP of the HA-tagged JMJD3 protein. Cells were grown in SILAC medium for 5 passages and were validated for amino acid incorporation before the experiment. We did not detect major differences in JMJD3 protein interactors, when JMJD3 was translated from mRNA templates with or without its 3′UTR. To investigate if the KDM6B 3′UTR controls JMJD3 activity through differences in protein folding, we performed XL-MS in cells expressing HA-tagged JMJD3 from an mRNA template with or without its 3′UTR. JMJD3 has a long intrinsically disordered region (IDR), containing more than 1100 amino acids and a structured JmjC domain at the C-terminus. Only in the presence of the long IDR is the activity of JMJD3 protein dependent on the 3′UTR. XL-MS revealed that translation of JMJD3 from an mRNA template with the 3′UTR changes co-translational protein folding in cells. When the protein was expressed in the presence of the 3′UTR, the majority of unique XL map to IDR-IDR interactions, whereas lack of the 3′UTR significantly increased the number of crosslinks involving the structured, enzymatic domain, which is consistent with reduced protein activity, likely caused by co-translational misfolding.