Herein we report the targeting effect of 1,2,3-triazolium salt derivatives of allobetulin on cancer cells mitochondria and their antiproliferative mechanism. A series of allobetulin derivatives with 1,2,3-triazolium positively charged units was designed and synthesized by multi-component triazolization reaction and alkylation. The screening of cytotoxicity showed that all the 1,2,3-triazolium salt derivatives of allobetulin displayed better cytotoxicity than the parent compound allobetulin and commercial anticancer drugs gefitinib. The most potent compound 4q showed strong anticancer activity, especially for Eca-109 cells. Compound 4n showed the strongest inhibitory effect on SGC-7901 cells. Further anticancer mechanism studies indicated that compounds 4n and 4q induced apoptosis through the mitochondrial pathway. Compounds 4n and 4q acted on mitochondria to cause an increase in intracellular reactive oxygen species and a change in the level of apoptosis-related protein (Bcl-2, Bcl-xL and Bax), which resulted in a decrease in membrane potential and activation of caspase family to induce cancer cells apoptosis. Meanwhile, compounds 4n and 4q could induce cancer cells apoptosis by arresting the cell cycle. Due to the strong cytotoxicity of compounds 4n and 4q, they are expected to become new anticancer agents and deserve further study.