AbstractToosendanin (TSN) is an active compound from the fruit of Melia toosendan Sieb et Zucc. TSN has been shown to have broad‐spectrum anti‐tumour activities in human cancers. However, there are still many gaps in the knowledge of TSN on canine mammary tumours (CMT). CMT‐U27 cells were used to select the optimal acting time and best concentration of TSN to initiate apoptosis. Cell proliferation, cell colony formation, cell migration and cell invasion were analysed. The expression of apoptosis‐related genes and proteins were also detected to explore the mechanism of action of TSN. A murine tumour model was established to detect the effect of TSN treatments. The results showed that TSN decreased cell viability of migration and invasion, altered CMT‐U27 cell morphology, and inhibited DNA synthesis. TSN‐induced cell apoptosis by upregulating BAX, cleaved caspase‐3, cleaved caspase‐9, p53 and cytochrome C (cytosolic) protein expression, and downregulating Bcl‐2 and cytochrome C (mitochondrial) expression. In addition, TSN increased the mRNA transcription levels of cytochrome C, p53 and BAX, and decreased the mRNA expression of Bcl‐2. Furthermore, TSN inhibited the growth of CMT xenografts by regulating the expression of genes and proteins activated by the mitochondrial apoptotic pathway. In conclusion, TSN effectively inhibited cell proliferation, migration and invasion activity, as well as induced CMT‐U27 cell apoptosis. The study provides a molecular basis for the development of clinical drugs and other therapeutic options.