The period circadian regulator 3 (PER3) has been reported to play a negative role in human immortalized bone marrow-derived Scp-1 cells (iBMSCs) and patient adipose-derived stromal cells (PASCs) or a negative/positive role in mice adipogenesis. However, human PER3 (hPER3) was identified as a positive regulator of human adipose tissue-derived stromal cells (hADSCs) adipogenesis in this study. Silencing or overexpression of hPER3 in hADSCs inhibited and promoted adipogenesis in vitro. In vivo, the overexpression of hPER3 increased high-fat diet-induced inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) forms, increasing systemic glucose intolerance and insulin resistance. Molecularly, hPER3 does not interact with hPPARγ, but represses Notch1 signaling pathway to enhance adipogenesis by interacting with hHSP90AA1, which is able to combine with the promoter of hNotch1 and inactivate its expression. Thus, our study revealed hPER3 as a critical positive regulator of hADSCs adipogenesis, which was different from the other types of cells, providing a critical role of it in treating obesity.