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Background: Genus Kalanchoe (Crassulaceae) comprises plants originating from Africa and Asia. Their pharmaceutical preparations are commercially available in the form of anti-inflammatory and antimicrobial medicaments. Recent studies have shown anticancer activity of Kalanchoe plants. However, studies on Kalanchoe blossfeldiana (KB) are extremely limited, and its cytotoxic properties have not yet been evaluated. Objective: The objective of the study is to estimate the cytotoxic activity of KB on human cervical carcinoma (HeLa) cells and determine the mode of cell death. Materials and Methods: The cytotoxic activity of ethanolic extract of leaves of KB was tested on HeLa cells using Real Time xCelligence System (RTCA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death, the generation of reactive oxygen species (ROS), depolarization of the mitochondria, cell cycle arrest, and the activity of caspases-3/7/9 were assessed by flow cytometry or a luminometer. The activity of poly (ADP-ribose) polymerase (PARP), translocation of apoptosis-inducing factor (AIF) in the cells, and expression of 92 genes associated with apoptosis/necrosis were also estimated. Results: According to the results, the IC50values of KB extract were 8.28 ± 0.29 and 9.63 ± 1.07 μg/mL in RTCA and MTT assay, respectively. The results showed a significant increase in the generation of ROS and depolarization of the mitochondria in the dead cell population. Caspases-3/7/9 were not activated during treatment with KB extract. We also found that the pathway of HeLa cell death was induced by tumor necrosis factor-related apoptosis-inducing ligand and was independent of the PARP and AIF. The extract also induced cell cycle arrest in S phase. Conclusions: The results of this study indicate that KB extract induces necrosis in HeLa cells and this process is death receptor-mediated leading to the overproduction of ROS, mitochondrial dysfunction, and cell cycle arrest. [ABSTRACT FROM AUTHOR]