We previously identified the human HMBOX1 (homeobox containing 1) gene, which represents a distinct group of HNF transcription factors, and is a potential transcription repressor with highly expression in pancreas. In our present work, we found that there exists a splicing variant of HMBOX1, designated HMBOX1b. Compared to HMBOX1, HMBOX1b encodes a 304 amino acids protein that shares the N-terminal region but has no homeodomain and the C-terminal region because of an alternative exon 7 which results in reading frame shifting. Unlike the highly pancreatic expression of HMBOX1, HMBOX1b was ubiquitous expressed in all human tissues detected by RT-PCR. Immunofluorescence staining showed that HMBOX1b accumulated in both cytoplasm and nucleus, and transcriptional reporter assays indicated that HMBOX1b only retained faint transcriptional repressive activity. Taken together, our findings suggest a distinct role of HMBOX1b, and the control of mRNA splicing might be involved in homeobox genes regulation.