Mlr1 (Mblk-1-related protein-1) and Mlr2 are mouse homologs of transcription factor Mblk-1 (Mushroom body large-type Kenyon cell-specific protein-1), which we originally identified from the honeybee brain. In the present study, aiming at identifying coregulator(s) of Mlr1 and Mlr2 from the mouse brain, we used yeast two-hybrid screening of mouse brain cDNA library to search for interaction partners of Mlr 1 and Mlr2, respectively. We identified nucleolar protein 4 (NOL4) splicing variants as major interaction partners for both Mlr1 and Mlr2. Among the three murine NOL4 splicing variants, we further characterized NOL4-S, which lacks an N-terminal part of NOL4-L, and NOL4-SΔ, which lacks nuclear localization signal (NLS)-containing domain of NOL4-S. A GST pull-down assay revealed that Mlr1 interacts with both NOL4-S and NOL4-SΔ, whereas Mlr2 interacts with NOL4-S, but not with NOL4-SΔ. These results indicate that the NLS-containing domain of NO4-S Is necessary for in vitro binding with Mlr2, but not for that with Mlr1. Furthermore, a luciferase assay using Schneider's Line 2 cells revealed that transactivation activity of Mlr1 was significantly suppressed by both NOL4-S and NOL4-SΔ, with almost complete suppression by NOL4-SΔ. In contrast, transactivation activity of Mlr2 was significantly suppressed by NOL4-S but rather activated by NOL4-SΔ. Our findings suggest that transactivation activities of Mlr1 and Mlr2 are differentially regulated by splicing variants of NOL4, which are expressed in a tissue-selective manner.