Transforming growth factor β (TGF‑β) is a polypeptide growth factor with various biological activities, and is widely distributed in various tissues. In mammals, TGF‑β has three isoforms: TGF‑β1, 2, and 3, of which TGF‑β1 is most abundant in the TGF‑β family. TGF‑β1 is closely related to the occurrence and development of tumors. A large number of previous studies have shown that melatonin can inhibit a variety of malignancies. Thus, the aim of the present study was to investigate the role of TGF‑β1 in the melatonin‑mediated inhibition of the proliferation of gastric cancer cells in vitro and in vivo. TGF‑β1 cytokine stimulation, anti‑TGF‑β1 neutralizing antibody blocking, siRNA TGF‑β1 and other means were utilized to explore the role of TGF‑β1 during the course of anti‑gastric cancer by melatonin. The results showed that melatonin upregulated the expression of TGF‑β1 in tumor tissues during the process of inhibiting gastric cancer tumor growth in vivo. Melatonin inhibited the proliferation of gastric cancer cells in vitro, accompanied by increased expression of TGF‑β1 in a time‑dependent manner. siRNA‑mediated silencing of TGF‑β1 and anti‑TGF‑β1 neutralizing antibody completely blocked the TGF‑β1 pathway, which significantly antagonized the melatonin‑mediated inhibition of the growth and proliferation of gastric cancer cells, and promoted G1 phase to S phase transformation of MFC cells. Our findings suggest that TGF‑β1 is involved in the regulation of the proliferation of tumor cells. One of the ways in which melatonin inhibits the proliferation of gastric cancer cells is dependent on the TGF‑β1 signaling pathway.