Six genes are homologs of Drosophila sine oculis and encode transcription factors that are characterized by a conserved Six domain and homeodomain. Of the six family members (Six1–Six6) in mice, Six1 and Six4 show similar expression patterns during embryogenesis. Six1–/– mice show defective formation of various organs such as inner ear, nose, skeletal muscle, kidney and thymus, whereas Six4–/– mice show little anomaly in organogenesis. To understand the molecular basis for the differential function of Six1 and Six4 in vivo, we screened target genes of Six1 and Six4 and found that Six1 and Six4 differentially regulated a set of target genes. Gel‐retardation assays indicated that the promoter region of one of the targets, sodium–potassium–chloride cotransporter 1 (Slc12a2), contains multiple Six1‐binding sites and one common binding site of Six1 and Six4, suggesting that the DNA‐binding specificity of Six1 is distinct from that of Six4. This underlies the differential regulation of common target genes by Six1 and Six4. Furthermore, in situ hybridization demonstrated that the expression of Slc12a2 was reduced in the developing dorsal root ganglia of Six1–/–/Six4–/– mice, suggesting that Six1 and Six4 regulate Slc12a2 in vivo.