Microtubule-interacting and trafficking domain containing 1 (MITD1) is associated with abscission during cytokinesis. However, systematic investigation into its role in cancer is lacking. Therefore, we explored the pan-cancer role of MITD1 using multiple databases. Expression and clinical survival, immunological, and enrichment analyses were performed using R packages and online tools. For breast cancer, single-cell level analysis, immunochemistry, and in vitro experiments were performed to explore the mechanism of MITD1. A nomogram was established to predict the prognosis of patients with breast cancer and evaluate the immunotherapy biomarker based on two datasets. In some cancers, high MITD1 expression was associated with a more favorable prognosis. For instance, it inhibited tumor cell proliferation and migration in breast cancer. MITD1 may regulate cancer development by altering the tumor microenvironment, and MITD1 expression may predict the response to immune checkpoint blockade, platinum, and poly ADP-ribose polymerase inhibitor therapies. Our nomogram was used to determine the prognosis of patients with breast cancer. MITD1 can also predict the response to immunotherapy. Our first pan-cancer study of MITD1 has shown that it plays different roles in cancer development and therapy. In breast cancer, MITD1 inhibited cell proliferation and migration and serves as a new biomarker.