11beta-hydroxylase deficiency (11beta-OHD) occurs in about 5-8% of congenital adrenal hyperplasia (CAH). In this study, we identified three CYP11B1 (encoding Cytochrome P450 11B1) heterozygous mutations: c.1358G>C (p.R453Q), c.1229T>G (p.L410R) and c.1231G>T (p.G411C) in a Chinese CAH patient due to classic 11beta-OHD. His parents were healthy and respectively carried the prevalent mutation c.1358G>C (p.R453Q), and the two novel mutations c.1229T>G (p.L410R) and c.1231G>T (p.G411C). In vitro expression studies, immunofluorescence demonstrated that wild type and mutant (L410R and G411C) proteins of CYP11B1 were correctly expressed on the mitochondria, and enzyme activity assay revealed the mutant reduced the 11-hydroxylase activity to 10% (P<0.001) for the conversion of 11beta-deoxycortisol to cortisol. Subsequently, three dimensional homology models for the normal and mutant proteins were built by using the x-ray structure of the human CYP11B2 as a template. Interestingly, in the heme binding site I helix, a change from helix to loop in four amino acide took place in the mutant model. In conclusion, this study expands the spectrum of mutations in CYP11B1 causing to 11beta-OHD and provides evidence for prenatal diagnosis and genetic counseling. In addition, our results confirm the two novel CYP11B1 mutations led to impaired 11-hydroxylase activity in vitro.