Wogonin (WOG) has been demonstrated to have anti-cancer activity, but the mechanisms remain unclear. In this study, new targets of WOG were predicted for lung cancer, bladder cancer, and colon cancer by using bioinformatics methods. WOG might primarily suppress cancers via regulating arachidonic acid, Ras, MAPK, linoleic acid, PI3K-Akt, and folate biosynthesis pathways. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay showed that WOG inhibited the proliferation of A549 cells. Real-time quantitative reverse transcription PCR (RT-qPCR) results indicated that anti-lung cancer effect of WOG was achieved by regulating the expression of 18 target genes, including AKR1B10, AKR1C3, BDNF, CAV1, CXCL2, CYP2B6, CYP4F3, DAO, EGF, ENO3, IL6, PLA2G1B, PLA2G2F, PLA2G4A, PTGES, SLCO1B1, SLCO1B3, and TFAP2A. The Kaplan-Meier survival curves further confirmed that DAO, PLA2G1B, SLCO1B3 and TFAP2A were essential targets via which WOG affected lung cancer survival. Moreover, BDNF, FGF2, and PTGS1 were predicted to be the targets via which WOG alleviated cancer proliferation and invasion in bladder cancer. As for colon cancer, WOG might induce autophagy and inhibit proliferation by down-regulating NTF4 and TH. The study will provide clue for using WOG as a promising antineoplastic agent in basic and translational research, and bring light to the application of herbs containing WOG as food supplements.