Odd-skipped related 2 (Osr2) is expressed in the domain of epithelial-mesenchymal interactions during tooth and kidney development. In this study, we report that alternative splicing of exon 4 resulted in two transcripts, Osr2A and Osr2B. These variants utilized a different splice acceptor, and Osr2B exhibited a frameshift followed by an early stop codon. Osr2A mRNA produced a protein that was 36 amino acids longer than Osr2B at the C-terminus and had five zinc-finger domains, whereas Osr2B had three zinc-finger motives. The 2 Osr2 variants were found in kidney, skeletal muscle, and testis, and Osr2B was predominantly transcribed in each. Flag-tagged variants showed the same localization in cell nuclei, however, Gal4 fusion proteins showed opposite transcriptional activities. Further, Flag-tagged variants also showed opposite transcriptional activities, though they were in contrast to the findings for the Gal4-fused variants. Our results indicate that Osr2 bifurcates its function by alternative splicing.