Intermittent fasting as an adjuvant therapy in clinical practice is an emerging treatment modality to target tumor growth by reducing glucose utilization. Berberine, an alkaloid extracted from the traditional Chinese medicine Coptidis Rhizoma, has been shown to be a safe and effective antitumor agent in several cancers. Hence, the purpose of the present study was to investigate the effects of the combination of berberine and low glucose on gastric cancer. Our results showed that the combination of berberine and low glucose effectively inhibited cell viability, promoted apoptosis, and reduced the migration ability of MGC803 cells. In addition, the combination was shown to activate the PP2A/GSK38 signaling axis, leading to the downregulation of the downstream prosurvival protein MCL-1, which leads to the death of gastric cancer cells. In addition, the inhibitor of GSK38 partially reversed the effect of this combination on MGC803 cells. In vivo experiments demonstrated that berberine effectively impaired the growth of xenograft tumors, when administered during intermittent fasting (hypoglycemic conditions), and was well tolerated by nude mice without the occurrence of any adverse effects. Based on these results, we conclude that the berberine/low-glucose combination can inhibit the growth of gastric cancer through the PP2A/GSK38/MCL-1 signaling pathway. Accordingly, this combination of drugs and lifestyle may become a new type of safe and effective anti-cancer therapy.