Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that mediates apoptosis signaling. To investigate the possible role of ASK1 in regulating E2F1, we examined the effect of ASK1 on transcriptional activity of E2F1. E2F1 is known as a transcription factor which induces several proteins involved in cell cycle, especially in G1/S transition. This study presents the biochemical and functional interactions between ASK1 and E2F1. ASK1 represses E2F1 transcriptional activity. Data further show that the stability of E2F1 by ASK1 doesn’t be affected and kinase activity of ASK1 is not crucial for E2F1’s activity. C-terminal deletion mutant of ASK1 is unable to repress the transcriptional activity of E2F1. Moreover, in vivo binding result shows an interaction between ASK1 and E2F1 and the C-terminal region of ASK1 interacts with E2F1. Therefore, we conclude that ASK1-mediated E2F1 suppression is caused by interaction between ASK1 and E2F1. Based on our data, this study proposes that ASK1 plays an important role in regulating E2F1 transcriptional activity as well as in regulating apoptosis.