A series of tryptamine salicylic acid derivatives were synthesized and their antiproliferative activity against MGC-803, MCF-7, HepG2, A549 and HeLa cell lines was evaluated. The structure-activity relationship (SAR) study revealed that different substitutions of the C5 and C3'-C5' positions have certain effects on the anti-proliferation activity. The growth assay revealed that <b><i>N</i>-[2-(5-bromo-1<i>H</i>-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-benzamide</b> (<b>E20</b>) showed the most potent and broad-spectrum anticancer inhibition of all the cell lines evaluated, and was only more potent than 5-Fu for the gastric cancer cell line. Preliminary studies indicated that compound <b>E20</b> could inhibit colony formation and migration of MGC-803 cells. The flow cytometry (FCM) results showed that compound <b>E20</b> arrested the cell cycle in the G2/M phase and induced apoptosis of MGC-803 cells in a concentration-dependent manner. In addition, the western blot results showed that <b>E20</b> can down-regulate the expression of hexokinase 2. Our studies suggest that the framework of <b><i>N</i>-[2-(5-bromo-1<i>H</i>-indol-3-yl)-ethyl]-2-hydroxy-3-methyl-benzamide</b> may be consider as a new type of chemical for designing effective anti-cancer drugs targeting gastric cancer cells.