Fibroblast growth factor receptor 2 (FGFR2) plays an important regulatory role in bone development. However, the regulatory mechanisms controlling FGFR2 expression remain poorly understood. Here we have identified a role for the nuclear factor Y (NF-Y) in constitutive activation of FGFR2. A unique DNase I hypersensitive site was detected in the region encompassing nucleotides -270 to +230 after scanning a large range covering 33.3 kilobases around the transcription start site of FGFR2. Using a PCR-based chromatin accessibility assay, an open chromatin conformation was detected around the proximal 5' fragment of FGFR2 gene. Deletion constructs of the 5'-flanking region of FGFR2 were fused to a luciferase reporter gene. After transient transfection in C3H10T1/2, ME3T3-E1, and C2C12 as well as primary osteoblasts, a minimal region -86/+139 that is highly homologous to the human sequence and bears a CCAAT box was identified as the core promoter. Electrophoretic mobility shift assay supershift and chromatin immunoprecipitation demonstrated that the CCAAT box was the binding site for NF-Y. Deletion of NF-Y consensus sequence resulted in the total loss of NF-Y promoter activity. Overexpression of NF-Y protein and transfection of NF-Y small interfering RNAs in the cells substantially changed the promoter activity. Moreover, NF-Y small interfering RNAs greatly inhibited the endogenous FGFR2 transcription level and the chromatin accessibility and H3 acetylation across the promoter. Taken together, our results demonstrate that interaction of NF-Y at the CCAAT box is pivotal to FGFR2 gene transcription partly through the construction of a local open chromatin configuration across the promoter.