The release of lysosomal hydrolase into the cytoplasm is accompanied by several systems of apoptosis signal transduction, and the imbalance between cell viability and apoptosis induces tumorigenesis. Tea polysaccharides (TPs) are the main bioactive components in green tea with hopeful anti-tumor efficacy, while their mechanism is still unclear. Here, TPs significantly promoted the death of colon cancer cell line CT26. RNA-seq results showed that the signal pathways up-regulated by TPs included lysosome pathways, apoptosis, the release of mitochondrial pigment c and programmed cell death. Among them, the results of AO-EB and annexin V-FITC/PI double staining indicated that TPs significantly up-regulated apoptosis. In addition, TPs significantly disrupted the function of lysosomes, which would cause mitochondrial damage. Intriguingly, TPs treatment increased the expression of Bak1, cleaved caspase-9 and cleaved caspase-3, but decreased the level of Bcl-2 and mitochondrial membrane potential, which indicated that TPs induced mitochondrial-mediated apoptosis. Moreover, TPs ameliorated the reduced lysosomal numbers by Baf A1 (lysosomal inhibitor). Therefore, our data indicated that TPs targeted lysosomes and induced apoptosis by a lysosomal-mitochondrial pathway mediated caspase cascade, thereby inhibiting the proliferation of CT26 cells. In short, the data would help the development of TPs as potential cancer drug therapeutics.