The development of novel, safe, and effective anti-tumor agents represents an essential strategy within cancer chemotherapy. In the present study, a total of 60 flavone and chalcone derivatives containing benzimidazole groups were synthesized. The anti-proliferation tests revealed that most of the benzimidazole-chalcone derivatives exhibited excellent inhibitory activity against various tumor cell lines, while the benzimidazole-flavone derivatives showed relatively weak effects. Among the benzimidazole-chalcone derivatives, compound 19o with potential as a candidate anti-tumor drug was selected, and its anti-tumor biological activities in vitro and in vivo were evaluated. In the gastric cancer cell lines with superior anti-proliferation activity, compound 19o showed IC ₅₀ values of less than 20 μM in MGC-803, SGC-7901 and HGC-27 cells, and it exhibited certain selectivity when compared with normal GES-1 cells. Compound 19o was verified to have inhibitory effects on colony formation and promote apoptosis in HGC-27 and SGC-7901 cells, and can block the cycle of HGC-27 cells in the G0/G1 phase. Further evaluation revealed that compound 19o inhibits the glycolytic pathway by suppressing the expression of HIF-1α and the key rate-limiting enzymes HK2 and PKM2 in glycolysis, and reduces the intracellular lactate content. In the HGC-27 xenograft nude mice, compound 19o was able to significantly inhibit the growth of tumors, and the inhibition rates at doses of 15 mg/kg and 30 mg/kg were 30.95 % and 62.89 %, respectively. Furthermore, the compound 19o demonstrated certain safety in vivo , and exhibited acceptable pharmacokinetic properties after intravenous administration. Overall, the benzimidazole chalcone derivative 19o synthesized and screened in this study is a potential candidate drug for anti-tumor treatment.