Objective To establish primary culture，expansion，and identification methods for rat inferior vena cava (IVC)-derived mesenechymal stem cells(MSCs)，providing a novel alternative source. Methods Rat IVC-MSCs were isolated using mechanical fragmentation combined with enzymatic digestion. Multidimensional characterization included morphological observation，flow cytometry for surface immunophenotyping，and trilineage differentiation (osteogenic，adipogenic and chondrogenic) induction with histological staining. Results Primary cells exhibited short spindle morphology with adherent growth and robust proliferation. At passage 4，marker protein expression: CD90 （97.57 ± 2.83）%、CD73 （99.67 ± 0.06）%、CD44 （99.53 ± 0.06）%，positive rate of positive markers > 95%；CD45 （3.24 ± 0.19）%、CD34 （3.82 ± 0.31）%、CD11b/c （4.88 ± 0.24）%，positive rate of negative markers <5%. Induced cells successfully differentiated into osteoblasts，adipocytes，and chondrocytes，demonstrating multipotency. Conclusion We successfully established a rat IVC-MSC culture system，expanding the MSC“seed bank” to this uncommon source. This broadens MSC acquisition avenues and establishes a reproducible platform for investigating biological characteristics, elucidating venous homeostasis mechanisms, and developing stem cell therapies for vascular diseases，providing a paradigm for clinical translation of stem cells from rare tissue sources.