Ebp1, an ErbB3 receptor-binding protein, inhibits cell proliferation and acts as a putative tumor suppressor. Ebp1 translocates into the nucleus and functions as a transcription co-repressor for E2F-1. Here, we show that Ebp1 p42 isoform can be sumoylated on both K93 and K298 residues, which mediate its nuclear translocation and are required for its anti-proliferative activity. We find that translocation in liposarcoma (TLS)/FUS, an RNA-binding nuclear protein that is involved in pre-mRNA processing and nucleocytoplasmic shuttling, has Sumo1 E3 ligase activity for Ebp1 p42. Ebp1 directly binds TLS/FUS, which is regulated by genotoxic stress-triggered phosphorylation on Ebp1. Ebp1 sumoylation facilitates its nucleolar distribution and protein stability. Overexpression of TLS enhances Ebp1 sumoylation, whereas depletion of TLS abolishes Ebp1 sumoylation. Moreover, unsumoylated Ebp1 mutants fail to suppress E2F-1-regulated transcription, resulting in loss of its anti-proliferation activity. Hence, TLS-mediated sumoylation is required for Ebp1 transcriptional repressive activity.