Histone H2B ubiquitylation has been implicated in transcriptional regulation. In humans, RING-finger domain type E3 ubiquitin ligase RNF20/40 catalyzes monoubiquitylation of histone H2B and participates in various biological processes. However, it is uncertain that all these RNF20/40-related cellular events are only mediated by H2B ubiquitylation. Several researches suggest that some of these events might be due to additional substrates of RNF20/40. In order to identify unknown non-histone substrates of RNF20/40, we tried immunoprecipitation using FLAG-RNF20 cell line to screen RNF20/40-interacting proteins. Coimmunoprecipitated proteins were identified by mass spectrometry analysis. We found that isoform 2 of elongation factor 1-delta, eEF1deltaL, directly interacts with RNF20/40 and is ubiquitylated by RNF20/40. We show that function of eEF1deltaL as a heat shock response transcription factor is facilitated by RNF20/40.