In mammalian cells, the RING-finger type E3 ubiquitin ligase RNF20/40 catalyzes mono-ubiquitylation at lysine 120 of histone H2B that is implicated in transcriptional regulation. Previous studies have shown that depletion of RNF20/40 causes defects in various cellular events, suggesting that RNF20/40 may have additional ubiquitylation target proteins other than histone H2B. In order to find novel non-histone target proteins of RNF20/40, we performed co-immunoprecipitation using FLAG-RNF20 cell line. Subsequent mass spectrometry analysis identified eEF1BδL, a DNA binding transcription factor that is previously known to regulate expression of heat shock responsive genes, as an RNF20/40-interacting protein. In an in vitro ubiqtuiatylaion assay using purified factors, we found that eEF1BδL can be ubiquitylated by RNF20/40 and RAD6-dependent manner. In addition, we further found that RNF20/40-dependent ubiquitylation of eEF1BδL enhances expression of heat shock responsive genes with underlying mechanism that involves a cooperative interaction of eEF1BδL and RNF20/40 with HSF1. Collectively, our study demonstrates a novel transcriptional regulation pathway of heat shock responsive genes via ubiquitylation of eEF1BδL by RNF20/40.