The effect of 5-heptadecylresorcinol (AR-C17) on human breast cancer MDA-MB-231 cells and its underlying mechanisms were investigated. CCK-8 assay was used to detect the change of cell viability under different treatments. DCF-DA fluorescence probe assay was used to detect reactive oxygen species (ROS) changes. Flow cytometry was applied to measure the expression of intracellular tumor proliferation marker Ki-67, cell apoptosis changes, mitochondrial potential changes and autophagosome formation. Western blotting was used to detect the expression of related proteins including Bax, Bcl-2, LC3-Ⅱ. Compared with control group, AR-C17 treatment significantly decreased MDA-MB-231 cells viability and the expression of Ki-67, a marker of tumor proliferation. Furthermore, AR-C17 treatment could induce cells apoptosis by inducing intracellular ROS, reducing mitochondrial membrane potential, upregulating Bax/Bcl-2 ratio as well as increasing actived Caspase. Meanwhile, AR-C17 activated autophagy by increasing LC3-Ⅱ expression level and autophagosomes formation in cytoplasm. Inhibition of autophagy could further reduce cell viability and enhanced AR-C17-induced cell apoptosis. AR-C17 could inhibit proliferation of human triple-negative breast cancer MDA-MB-231 cells through increasing cell apoptosis and autophagy.