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The eukaryotic Mediator, comprising a large Core (cMED) and a dissociable CDK8 kinase module (CKM), regulates RNA Polymerase II (Pol II)-dependent transcription. cMED recruits Pol II and promotes pre-initiation complex (PIC) formation in a manner inhibited by the CKM, which is also implicated in post-initiation control of gene expression. Herein we report cryo-electron microscopy structures of the human complete Mediator and its CKM, which explains the basis for CKM inhibition of cMED-activated transcription. The CKM binds to cMED through an intrinsically disordered region (IDR) in MED13 and HEAT repeats in MED12. The CKM inhibits transcription by allocating its MED13 IDR to occlude binding of Pol II and MED26 to cMED and further obstructing cMED-PIC assembly through steric hindrance with TFIIH and the +1 nucleosome. Notably, MED12 binds to the cMED Hook, positioning CDK8 downstream of the transcription start site, which sheds new light on its stimulatory function in post-initiation events.