Intercellular signalling communication between adipose and muscle tissue has been investigated. To test the effect of muscle cells on adipogenic gene expression, we utilised an in vitro co-culture system, in which fat (3T3-L1) and muscle (L-6) cells were physically separated but chemically exposed each other via insert with 0.4 µm porous membrane. When 3T3-L1 and L-6 cells reached at 80 and 40% confluence, respectively in separate wells, L-6 cells grown in insert were transferred onto 6-well plates where 3T3-L1 cells were being grown. When both cells were fully differentiated in co-culture plates, morphology of 3T3-L1 was examined by staining with Oil-red-O. Activity of glycerol-3-phosphate dehydrogenase (GPDH) and adipogenic gene expression including lipoprotein lipase (LPL), adipsin, GPDH, peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer binding protein (C/EBPα) were analysed. The presence of muscle cells during preadipocyte differentiation inhibited (P < 0.05) lipogenesis by suppressing lipogenic gene expression including LPL, adipsin and GPDH. Furthermore, GPDH activity was also decreased (P < 0.05) in 3T3-L1 cells by the presence of L-6 cells. These results suggest that presence of muscle cells suppresses adipogenic differentiation by inhibiting the adipogenic gene expression and GPDH activity in the muscle and fat cell co-culture system.