Odd-skipped related 2 (Osr2) gene is mouse homolog of Drosophila Odd-skipped gene involved with the pair-rule segmentation phenotype in Drosophila mutant embryos. In this study, to examine Osr2 expression regulation, the mouse Osr2 promoter region was cloned and characterized, and found to have two enhancer elements in the -1463/-1031 (distal) and -581/+3 (proximal) regions, and a repressor region (-4845/-1463, far distal). CCAAT/enhancer binding protein (C/EBP) binding sites were found in both the distal and proximal enhancer elements. Osr2 promoter activity was enhanced by C/EBPdelta, a member of the C/EBP family, in a dose-dependent manner. Electrophoresis mobility shift assays showed that purified GST-C/EBPdelta bound to distal (-1295/-1261) and proximal (-89/-55) C/EBP binding motifs. Chromatin immunoprecipitation demonstrated that acetylated histones H3, H4, and C/EBPdelta in the proximal region (-280/-43), but not the distal region (-1438/-1196), indicating that the Osr2 promoter proximal region was transcriptionally activated in C3H10T1/2 cells. Our results suggest that Osr2 expression is regulated by C/EBP regulatory elements.