Previous in vitro studies found that NFE2L1 was involved in mediating ascorbic acid-induced osterix expression and osteoblast differentiation via binding to the antioxidant response element of the osterix promoter. To test the role of NFE2L1 in regulating bone formation in vivo, we disrupted NFE2L1 specifically in osteoblasts. Mice expressing Cre under the control of Col12 promoter were crossed with NFE2L1 loxP mice to generate Cre+ knockout (KO) and Cre- wild type (WT) mice. Skeletal measurements by DEXA revealed 8-10% and 9-11% reduction in total body BMC and bone area in the KO mice from 3 to 8 weeks of age. pQCT analyses found both periosteal and endosteal circumferences were reduced by 6% at the mid diaphysis of the femurs from 8 week old KO mice. Histomorphometric analyses revealed reduced bone formation was a cause for reduced bone size in the KO mice. μCT analysis of the metaphysis of the femur revealed that trabecular bone volume/total volume, and trabecular numbers were decreased by 30% and 53% in the NFE2L1 KO mice. Expression of osterix was decreased by 57% in the bones of NFE2L1 KO mice. In vitro nodule assay found that mineralized nodule area was reduced by 68% in the cultures of bone marrow stromal cells from NFE2L1 KO mice. Treatment of primary osteoblasts with ascorbic acid increased osterix expression by 4-fold whereas loss of NFE2L1 in osteoblasts diminished ascorbic acid stimulation of osterix expression by 50%. Our data provide the first in vivo experimental evidence that NFE2L1 produced by osteoblasts is involved in regulating osterix expression, osteoblast differentiation and bone formation.