In mammalian cells, the RING-finger type E3 ubiquitin ligase RNF20/40 catalyzes mono-ubiquitylation of histone H2B that has been implicated in transcriptional regulation. RNF20 and RNF40 knockdown studies have shown defects in various cellular processes, suggesting presence of additional ubiquitylation target proteins of RNF20/40. In order to find novel non-histone target proteins of RNF20/40, we performed co-immunoprecipitation using FLAG-RNF20 cell line and subsequent mass spectrometry analysis identified eEF1BδL, the isoform 2 of elongation factor 1-delta, as an RNF20/40-interacting protein. We confirmed direct protein interaction between RNF20/40 and eEF1BδL and also found that eEF1BδL can be ubiquitylated by RNF20/40. In addition, we further showed that eEF1BδL ubiquitylation enhances expression of heat shock responsive genes. Our study demonstrates that RNF20/40 regulates gene expression through ubiquitylation of transcription factor in addition to histone H2B.