We have established a novel preadipocyte cell line from mouse adult mature adipocytes. The mature adipocytes were isolated from fat tissues by taking only the floating population of mature fat cells. The isolated mature adipocytes were de-differentiated into fibroblast-like cells. The in vitro studies showed that the cells could re-differentiate into mature adipocytes after over 20 passages. The in vivo transplantation study also demonstrated that the cells had the full potential to differentiate into mature adipocytes, which has not been shown for the 3T3-L1 preadipocyte cell line derived from mouse embryo. We have further analyzed the expression profile of key fat regulatory genes such as the peroxisome proliferator-activated receptorgamma or CCAAT/enhancer-binding protein gene families. We conclude that our cell line could be used as a preferred alternative to 3T3-L1, potentially reflecting the characteristics of mature adipocytes more, since the cell line is actually derived from adult mature adipocytes.