Bone morphogenetic proteins (BMPs) are implicated in cell-fate determination of embryonic stem (ES) cells and cancer cells. GREM1 (CKTSF1B1 or DAND2) and CER1 (Cerberus 1 or DAND4) are cysteine knot superfamily proteins, functioning as secreted-type BMP antagonists. BMP4 is preferentially expressed in diffuse-type gastric cancer cells. Here, vertebrate BMP4 orthologs were identified and characterized by using bioinformatics for comparative proteomics and comparative genomics analyses. Baboon BMP4 gene within AC153751.2 genome sequence encoded a 408-aa protein, showing A152V and S298P amino-acid substitutions compared with human BMP4. Cow Bmp4, bat Bmp4 and zebrafish bmp4 genes were located within AC149774.2, AC156788.2 and CR391996.2 genome sequences, respectively. Human BMP4 showed 99.5%, 98.0%, 97.8%, 97.1%, 96.3%, 83.3% and 71.1% total-amino-acid identity with baboon BMP4, cow Bmp4, bat Bmp4, mouse Bmp4, rat Bmp4, chicken bmp4 and zebrafish bmp4, respectively. Human BMP4 gene was found consisting of six exons, including novel exon 1C, and known exons 1 (1A or I), 1B (II), 2 (III), 3 (IV) and 4 (V). Forty human BMP4 ESTs started from exon 1, seven from intron 1 (5'-flanking region of exon 2), and two from exon 1C. Fourteen mouse Bmp4 ESTs started from exon 1, and one from intron 1. The 5'-flanking region of exon 1 and exon 1 itself, but not exons 1C and 1B, were well conserved between human BMP4 and rodent Bmp4 genes. The major promoter region of human BMP4 and rodent Bmp4 genes were located within the 5'-flanking region of exon 1. FOXA2, OLF1, and MYC-binding sites were conserved among the major promoter region of human, baboon, cow, bat, mouse and rat BMP4 orthologs.