Gastric cancer is a frequently occurring cancer with high mortality each year worldwide. Finding new and effective therapeutic strategy against human gastric cancer is still urgently required. Ginkgolic acid (GA), a botanical drug, is extracted from the seed coat of Ginkgo biloba L. with various bioactive properties, including anti-tumor. Unfortunately, if GA has antitumor effect on human gastric cancer and the underlying molecular mechanisms have yet to be investigated. In the present study, we found that GA markedly reduced the gastric cancer cell viability. Furthermore, GA treatment led to the reduced migration ability of gastric cancer cells, which was associated with the decreased protein expression levels of Rho-associated protein kinase 1 (ROCK1), matrix metalloproteinase-2 (MMP-2), MMP-9 and α-smooth muscle actin (α-SMA). In addition, GA dose-dependently induced apoptosis in gastric cancer cells through activating Caspase-9/-3 and poly(ADP-Ribose) polymerase (PARP), which was along with the reduced Bcl-2 and Bcl-xl expression levels, and the elevated Bax and Bad levels. Consistently, Cyto-c protein expression in cytoplasm was also up-regulated by GA. Moreover, the production of reactive oxygen species (ROS) was significantly induced by GA. The activation of signal transducer and activator of transcription 3/janus kinase 2 (Stat3/JAK2) signaling pathway was inhibited by GA treatment. Intriguingly, blocking Stat3/JAK2 activation could further promote apoptosis and reduce cell viability induced by GA. However, GA-induced cell death was clearly abolished by ROS scavenger NAC, while the activation of Stat3/JAK2 signaling was restored by NAC. In vivo, GA showed effective role in reducing gastric tumor growth. Together, the findings here indicated that GA could be considered as an effective therapeutic candidate against human gastric cancer progression in future.