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Our goal was to evaluate phytochemical characterization and the antitumor potential of Calotropis procera. The phytochemical constitution of the crude extract (CE) revealed the presence of flavonoids, glycosides and cardenolide. The MTT assay was used to evaluate the cytotoxicity of CE, methanolic (MF) and ethyl acetate fractions (EAF) of C. procera in canine osteosarcoma cells (OST), canine mammary tumor (CMT), and canine skin fibroblasts (non-tumor cell). Doxorubicin was also used as a positive control. Results showed that CE, MF and EAF promoted a decrease in the viability of OST and CMT cells and did not alter the fibroblasts viability. C. procera also decreased the number of cells, corroborating to the decrease in proliferation and the cell cycle arrest in the G0/G1 phase. It was also evaluated the cell morphology by light and fluorescence microscopy, being demonstrated a reduction in cytoplasmic and cell rounding characteristic of programmed cell death. Moreover, flow cytometry data demonstrated that CE treatment promoted increase of caspase-3 and p53, showing that the cell death was activated in OST cells. In addition, there was a decrease in CD31, VEGF, osteopontin and TGF-β after CE treatment, suggesting that CE exerts its antitumor effect by reducing angiogenesis and tumor progression in OST cells. Moreover, CMT cells showed a reduction in PCNA after treatment with MF and CE. Analyzing the data together, C. procera, especially CE, showed an antitumor potential in both OST and CMT cells, encouraging us to continue investigating its use in cancer therapy.