To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from β-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/α-smooth muscle actin or H-2b/α-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3±4.4% and 12.7±9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7±9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8±17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIα-positive ratio was 2.2±1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3±0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.