We previously established a novel cell line, termed HOZOT, derived from umbilical cord blood mononuclear cells that is characterized as a human cytotoxic regulatory T (Treg) cell line with a FOXP3+CD4+CD8+CD25+ phenotype. Here, we describe a new property of HOZOT cells: they actively penetrate into a variety of human cancer cell lines, but not into normal cell lines, and form apparent cell-in-cell structures. In the process of cell penetration, we observed that HOZOT cells adhered to target cells seemed to first insert their nuclei into the cytoplasm of target cells, distinct from the process of phagocytosis. In addition, blocking experiments showed that major histocompatibility complex class I is one of the target cell recognition molecules for HOZOT cells. Furthermore, we propose that cell-in-cell structures between HOZOT cells and target cancer cells could be one of the cytotoxic mechanisms of HOZOT cells.