Objective To extract spleen-derived mesenchymal stem cells (S-MSCs) from 7-week-old rats using enzymatic digestion, thereby establishing a standardized experimental foundation to address the technical gap in S-MSCs research in vitro, and to further investigate the splenic regenerative microenvironment and develop sMSC-based cell therapies for autoimmune diseases. Methods Spleens were aseptically isolated from 6-week-old SD rats, rinsed, minced, and digested with 0.1% type Ⅳ collagenase and 0.125% Trypsin for 60 minutes for primary S-MSCs culture. The target cells were identified through morphological observation, detection of surface CD markers, and adipogenic/osteogenic differentiation induction assays. Results Within 4~8 days of primary culture, adherent short spindle-shaped cells reached confluence, exhibiting vortex-like dense arrangements. Flow cytometry analysis of passage 4 cells showed high expression of CD90, CD73, CD44, and CD29, and low expression of CD34, CD45, and CD11b/c. Both adipogenic and osteogenic differentiation assays yielded positive results. Conclusion Enzymatic digestion can successfully isolate primary rat S-MSCs.