BACKGROUND: Gastrointestinal stromal tumors (GISTs) are a type of tumor that originates from gastrointestinal mesenchymal tissue. Although several somatic or germline mutation GIST mice were established, however, there is still a lack of an authentic mice GIST cell lines for further experimental study. METHODS: We developed a chemically induced C57BL/6 J GIST model using 3- methylcholanthrene. Tumor characteristics were confirmed through histology and IHC. Primary cells were isolated to establish the mGSTc01 cell line, and molecular profiling was conducted. Additionally, we established GIST model in immunocompetent mice to evaluate their sensitivity to imatinib. RESULTS: Our study successfully developed a chemically induced murine GIST model, characterized by positive staining of c-kit and DOG-1. The mGSTc01 monoclonal cell line exhibited slender morphology and expressed the c-kit marker, Whole exome sequencing uncovered mutations of Lamb1, MMP9, and c-kit in GIST cells and provided a detailed picture of the entire genome's copy number variations. RNA sequencing indicated genes associated with cell adhesion and focal adhesion were enriched in mGSTc01 cells. The mGSTc01 cells demonstrated obvious malignant behaviors, notably elevated migration, adhesion, and proliferation. In immunocompetent mice, subcutaneous xenografts not only reserved the aggressive phenotype but also displayed a response to imatinib, underscoring the model's applicability for advancing therapeutic research. CONCLUSION: We firstly established a mGSTc01 cell line derived from C57BL/6 J mice GIST tumor offers, which closely mimicking human disease characteristics. It is a potent platform for investigating tumor microenvironment of GIST in mice model, and provides a novel way for new therapeutic discoveries in GIST.