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Background: Breast cancer seriously affects physical and mental health of women. Despite advances in the clinical use of different treatments, breast cancer remains a major cause of mortality. Therefore, it is imperative to identify promising treatment options. In the present study, we investigated the effects of shikonin on 4T1 breast cancer cells and its potential mechanisms of action. Methods: BALB/c-derived mouse breast cancer 4T1 is very close to human breast cancer in growth characteristics and systemic response, so 4T1 cells were selected for further experiments. Cell viability, apoptosis, intracellular reactive oxygen species (ROS), mitochondrial activity, and cellular calreticulin (CRT) exposure were assessed to evaluate the antitumor effects and mechanisms of shikonin in vitro. Orthotopic tumor growth inhibition and splenic immune cell regulation by shikonin were evaluated in 4T1 breast cancer orthotopic mice in vivo. Results: In vitro, shikonin could inhibit cell proliferation, cause apoptosis, disrupt mitochondrial activity, and induce ROS production and CRT exposure. In vivo, shikonin inhibited tumor growth, increased the proportion of CD8+ T cells, and reduced the proportion of regulatory cells (CD25+ Foxp3+ T cells) in the spleen. Conclusions: Shikonin inhibits the growth of 4T1 breast cancer cells by disrupting mitochondrial activity, promoting oxidative stress, and regulating immune function. [ABSTRACT FROM AUTHOR]