Natural medicine has multi-levels, multi-paths and multi-targets, and an increasing number of reports have confirmed that the combination of natural medicine with chemotherapy drugs exhibit a significant synergistic effect. It is necessary to find drug combination strategies to enhance efficacy and reduce toxicity, which can relieve the restrictions on the use of several chemotherapy drugs that have serious toxicity. Our previous reports showed that DT-13 inhibits cancer proliferation, invasion, migration, metastasis, and angiogenesis and induces autophagy. In this study, we evaluated the anti-proliferation effect of DT-13 on a panel of 40 different cancer cell lines for the first time. Moreover, it is also the first time that the combination of DT-13 with 5 different chemotherapy drugs on 3 common cancer cells has been examined. We further confirmed that DT-13 enhanced the sensitivity of gastric cancer cells to topotecan (TPT) via cell cycle arrest in vitro and in vivo. Considering that TPT has been subjected to restriction because of its serious toxicity, DT-13 showed the ability to enhance its effect and reduce its toxicity, which could provide a strategy to reduce the toxic and clinical side effects of TPT.