The cleft palate only (CPO) is a common congenital defect with complex etiology in humans. The molecular etiology of the CPO remains unknown. Here, we report a loss-of-function mutation in X-linked TBX22 gene (T-box 22) in a six-generation family of the CPO with obvious phenotypes of both cleft palate and hyper-nasal speech. We identify a functional -73G>A mutation in the promoter of TBX22, which is located at the core-binding site of transcription factor ETS-1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1). Phylogenetic analysis showed that the sequence around the -73G>A mutation site is specific in primates. The mutation was detected in all five affected male members cosegregating with the affected phenotype and heterozygote occurred only in some unaffected females of the family, suggesting an X-linked transmission of the mutation in the family. The -73G>A variant is a novel single nucleotide mutation. Cell co-transfections indicated that ETS-1 could activate the TBX22 promoter. Moreover, EMSA and ChIP assays demonstrated that the allele A disrupts the binding site of ETS-1, thus markedly decreases the activity of the TBX22 promoter, which is likely to lead to the birth defect of the CPO without ankyloglossia. These results suggest that a loss-of-function mutation in the X-linked TBX22 promoter may cause the cleft palate through disruption of TBX22-ETS-1 pathway.