Adipocyte differentiation is key to determining the number of adipocytes during the development of obesity. Recent studies have shown that growth differentiation factor-5 (GDF5) promotes brown adipogenesis, however its role in white adipogenesis is still uncertain. The aim of the present study was to investigate the effect of GDF5 on white adipogenesis using 3T3-L1 preadipocyte model. In the present study, GDF5 was found to be differentially regulated during adipocyte differentiation. GDF5 protein increased the differentiation of 3T3-L1 preadipocytes, especially when these cells were exposed to hormone cocktails without insulin. During adipogenesis, GDF5 enhanced the expression of genes related to adipocyte differentiation and caused cells to enter the S phase. Short-hairpin-RNA knockdown of GDF5 in 3T3-L1 cells was found to prevent adipogenesis induced by a standard hormone cocktail and to downregulate the expression of adipocyte genes and proteins, this impairment could be partly rescued by GDF5 protein. Collectively, these results suggest that GDF5 can promote progression of the cell-cycle and increase numbers of cells in S phase, GDF5 might play a critical role in 3T3-L1 preadipocyte differentiation.