RNF20/40 E3 ubiquitin ligase-mediated histone H2B monoubiquitylation plays important roles in many cellular processes, including transcriptional regulation. However, the multiple defects observed in RNF20-depleted cells suggest additional ubiquitylation targets of RNF20/40 beyond histone H2B. In this study, using biochemically defined assays employing purified factors and cell-based analyses, we demonstrate that RNF20/40, in conjunction with its cognate E2 ubiquitin-conjugating enzyme RAD6, monoubiquitylates lysine 381 of eEF1BL, a heat shock transcription factor. Notably, monoubiquitylation of eEF1BL increases eEF1BL accumulation and potentiates recruitment of p-TEFb to the promoter regions of heat shock-responsive genes, leading to enhanced transcription of these genes. We further demonstrate that cooperative physical interactions among eEF1BL, RNF20/40, and HSF1 synergistically promote expression of heat shock-responsive genes. In addition to identifying eEF1BL as a novel ubiquitylation target of RNF20/40 and elucidating its function, we provide a molecular mechanism for the cooperative function of distinct transcription factors in heat shock-responsive gene transcription.