LIM-domain-binding proteins (CLIM/NLI/Ldb) are nuclear cofactors for LIM homeodomain transcription factors (LIM-HDs) and LIM-only proteins (LMOs). The LIM-interaction domain (LID) of Ldb is located in the carboxy-terminal region and encoded by the last exon (exon 10) of Ldb genes. It is known that the mammalian CLIM1/Ldb2 gene has a splice isoform, named CLIM1b, lacking the LID. However, little is known about the nature of CLIM1b or the evolutionary conservation of this type of alternative splicing in amphibians and teleost fish. Here, we demonstrate that splice isoforms lacking the LID are also present in the Ldb1 genes of mammals, chick, and Xenopus, as well as in fish paralog Ldb4. All these splicing variations occur in intron 9 and exon 10. We observed that Ldb4b (splice isoform lacking LID) is localized in the nucleus when expressed in mammalian culture cells, and binds to Ldb4a (splice isoform containing LID) but not directly to LIM proteins. However, Ldb4b binds to LMO4 via Ldb4a when coexpressed in culture cells. We also found that mouse Ldb1b lacks the ability to activate protein 4.2 promoter, which is stimulated by LMO2 and Ldb1. These findings suggest that splice isoforms of Ldb lacking LID are potential regulators of Ldb function.