Obesity is a worldwide disease caused by the excessive proliferation of adipocytes. Multiple factors, including melatonin and physical loading, are involved in the control of obesity. Melatonin has been shown to induce apoptosis on preadipocytes while physical loading such as fluid shear stress (FSS) affects the proliferation and differentiation of adipocytes. Here, we studied the combined effects of melatonin and FSS on 3T3-L1 preadipocytes. For physical loading, preadipocytes were stimulated with a maximum dynamic fluid shear stress of 1 Pa at 1 Hz for 2 hours with/without melatonin. The experiment conditions were divided into four groups: (1) control, (2) 1 mM melatonin treatment, (3) FSS, and (4) combined 1 mM melatonin and FSS. All groups had a fixed duration time of 2 hours. ERK, p-ERK, COX-2, $C/EBP{\beta}$, $PPAR{\gamma}$, osteopontin, Bax, caspase-3 and caspase-8 proteins were assessed by Western blot analysis. GAPDH was used as a control. Results showed that combined melatonin and FSS treatment activated the ERK/MAPK pathway but not COX-2. Furthermore, combined melatonin and FSS treatment significantly decreased $C/EBP{\beta}$ and $PPAR{\gamma}$ compared to other groups. However, caspase-3 and caspase-8 did not result in significant changes. In summary, combined melatonin and FSS appears to have the potential to inhibit adipogenesis and treat obesity.