The EVI1 transcriptional repressor is critical to the normal development of a variety of tissues and participates in the progression of acute myeloid leukaemias. The repressor domain (Rp) was used to screen an adult human kidney yeast two-hybrid library and a novel binding partner designated ubiquitously expressed transcript (UXT) was isolated. Enforced expression of UXT in Evi1-expressing Rat1 fibroblasts suppresses cell transformation and UXT may therefore be a negative regulator of Evi1 biological activity. The Rp-binding site for UXT was determined and non-UXT-binding Evi1 mutants (Evi1Δ706–707) were developed which retain the ability to bind the corepressor mCtBP2. Evi1Δ706–707 transforms Rat1 fibroblasts, showing that the interaction is not essential for Evi1-mediated cell transformation. However, Evi1Δ706–707 produces an increased proportion of large colonies relative to wild-type, showing that endogenous UXT has an inhibitory effect on Evi1 biological activity. Exogenous UXT still suppresses Evi1Δ706–707-mediated cell transformation, indicating that it inhibits cell proliferation and/or survival by both Evi1-dependent and Evi1-independent mechanisms. These observations are consistent with the growth-suppressive function attributed to UXT in human prostate cancer. Our results show that UXT suppresses cell transformation and might mediate this function by interaction and inhibition of the biological activity of cell proliferation and survival stimulatory factors like Evi1.