Exercise reduces the risk of breast cancer development and improves survival in breast cancer patients. However, the underlying mechanisms of this protective effect remain to be fully elucidated, and it is unclear whether exercise can attenuate the protumor effects of obesity and related hyperlipidemia on breast cancer growth and development. We hypothesized that exercise attenuates the negative effect of hyperlipidemia through normalization of the tumor microenvironment and improved T cell infiltrate. Hyperlipidemic ApoE mice with orthotopic EO771 breast tumors were randomly assigned to one of two voluntary running groups or sedentary controls, and muscular cytochrome c oxidase subunit IV (COX-IV) expression was used as a biomarker for the level of exercise. Tumors from mice with high muscular COX-IV expression took significantly longer to reach 100 mm ( P = 0.008), but showed no difference in growth rate once the tumor was established. Wheel running appeared to reduce internal metastases, but did not affect T cell infiltrate or the proportion of regulatory and cytotoxic T cells within the tumor. Serum levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased by tumor burden ( P = 0.02) and correlated with spleen weight ( P < 0.0001, R = 0.65). Furthermore, tumor hypoxia was significantly decreased in mice with high muscular COX-IV expression ( P = 0.01). Taken together, these results indicate that wheel running can slow the establishment of primary and secondary EO771 breast tumors and induce beneficial changes in the breast tumor microenvironment in ApoE mice. NEW & NOTEWORTHY In this first study to investigate the effect of exercise on tumor behavior in a hyperlipidemic model, we hypothesized that wheel running would counteract the protumorigenic environment generated by hyperlipidemia. Wheel running slowed establishment of primary and secondary tumors and reduced tumor hypoxia but did not affect exponential tumor growth in ApoE mice. Overall, voluntary wheel running induced favorable microenvironmental changes in breast tumors.