BACKGROUND Anorectal malformations (ARMs) represent a variety of congenital disorders that involve abnormal termination of the anorectum. This study was to reveal relation between CDX1 and human ARMs phenotypes. METHODS 108 Chinese patients and 120 Chinese controls were included in this study. We analyzed the relation between two by PCR, qRT-PCR, western blot and immunofluorescence. RESULTS Four heterozygous mutations in CDX1 gene were identified in ARMs patients (3.7%, 4/108), no found in controls. CDX1 protein expression was significantly decreased in the ARMs compared with the control anorectum. All samples analyzed in ARMs group exhibited down-regulated CDX1 mRNA expression in comparison to matched normal group, demonstrated significant differences statistically. CONCLUSIONS The findings represented the relation between CDX1 mutations and CDX1 genotype. Furthermore, it was suggested that the downregulation of CDX1 might be related to the development of ARMs.