Background <p>Mammary tumor is one of the most prevalent cancers in female companion dogs. Current chemotherapeutic treatments are often limited by high toxicity and financial burden, highlighting the need for safer and more accessible therapeutic alternatives.</p> Objective <p>This study aimed to evaluate the anticancer potential of the methanol extract of <i>Polytrichastrum alpinum</i>, a moss species native to Antarctica, in mammary tumor cell lines derived from companion dogs.</p> Methods <p>Two canine mammary tumor cell lines, MGT316 and MGT612, were treated with various concentrations of <i>Polytrichastrum alpinum</i> methanol extract. Cell viability, colony-forming ability, cell cycle distribution, apoptosis, migration, and invasion were assessed. Cytotoxic effects on normal canine epithelial cells were also evaluated to compare responses between tumor and non-tumorigenic cells.</p> Results <p><i>Polytrichastrum alpinum</i> methanol extract significantly reduced cell viability and colony-forming ability in a dose-dependent manner. It induced G0/G1 cell cycle arrest and promoted apoptosis in both tumor cell lines. Additionally, the extract effectively inhibited cell migration and invasion. Notably, minimal cytotoxic effects were observed in normal canine epithelial cells, indicating differential sensitivity between tumor and non-tumorigenic cells.</p> Conclusion <p>These findings demonstrate that <i>Polytrichastrum alpinum</i> methanol extract exerts antiproliferative, pro-apoptotic, and anti-invasive effects on mammary tumor cells of companion dogs, indicating its potential as a natural therapeutic agent. Furthermore, given the relevance of canine mammary tumors as a comparative model, this study may provide valuable translational insights for human breast cancer research within a One Health framework.</p>