Obesity is a major disease that causes significant complications. Inhibition of preadipocyte proliferation has the potential to prevent obesity and metabolic diseases. Melatonin is a pineal gland hormone that has various effects on cells and tissues. In this research, we investigated whether melatonin induces apoptosis in 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were cultured until confluence and then treated with 0, 10, 100, and 1000 μM melatonin for 1, 3, and 5 days. A cell viability assay kit was used for determining cell viability. Cell death marker proteins were assessed by Western blot analysis using GAPDH for control. Apoptotic morphological changes with nuclei fragmentation were observed using DAPI staining. Melatonin treatment decreased the phosphorylated extracellular signal-regulated kinases (p-ERK) activation while increasing the activation of caspase-3, 8, and 9. Furthermore, melatonin not only increased Bcl-2-associated X protein (Bax) but decreased B-cell lymphoma 2 (Bcl-2) expression as dose increases from 0 to 1000 μM. The melatonin treatment also suppressed the growth of preadipocytes with increasing concentration. These effects were attenuated by luzindole, a melatonin receptor antagonist and U0126, an inhibitor of p-ERK activation. In conclusion, melatonin can induce apoptosis of 3T3-L1 preadipocytes via p-ERK decrease.