As a gamma-secretase inhibitor, DAPT has been widely used to evaluate the biological behaviors and Notch signaling pathway in various cells. This study was aimed to examine the effects of DAPT on the growth and vitamin D(3) induced osteogenesis in adipose derived stem cells (ASCs). The cells were treated with or without DAPT and induced to osteoblastic lineage in the presence of vitamin D(3). Alizarin red staining and real-time PCR results indicated that the addition of DAPT to vitamin D(3) treatments enhanced osteogenesis in ASCs. According to the fold increase and colony-forming unit assay results, the cells cultured in DAPT exhibited lower proliferation rate than those cultured in control medium. Hey1, expressed in the nucleus of ASCs to act as a transcriptional repressor, was downregulated when Notch signaling was inhibited by DAPT. Whereas the expression of Runx2 increased in the nucleus of osteogenic induced ASCs after DAPT treatment. This study demonstrated that DAPT reduced the proliferation and enhanced the osteogenesis in ASCs via regulation of Notch and Runx2 expression.