It is well known that sex differences exist about the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of the osteoblast phenotypes of mouse osteoblasts, and then performed the comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts, compared to male. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression was the highest ratio of female to male. A reduction in endogenous levels of Serpina3n by siRNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells and receptor activator of nuclear factor kappaB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we first found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.