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Background: Annulus fibrosis (AF) defects have been identified as the primary cause of disc herniation relapse and subsequent disc degeneration following discectomy. Stem cell-based tissue engineering offers a promising approach for structural repair. Menstrual blood-derived mesenchymal stem cells (MenSCs), a type of adult stem cell, have gained attention as an appealing source for clinical applications due to their potential for structure regeneration, ease of acquisition, and low immunogenicity. Methods: The differential potential of MenSCs cocultured with AF cells was examined by the expression of collagen I, SCX, and CD146 using immunofluorescence. Western blot and ELISA were used to examine the expression of TGF-β and IGF-I in coculture system. An injectable gel containing MenSCs was fabricated and its repairment properties was evaluated in a discectomy animal model (Sprague-Dawley rats, males, 8 weeks old). Disc degeneration was assessed via magnetic resonance (MR) imaging and histological staining. Immunohistochemical analysis was performed to assess the expression of aggrecan, MMP13, TGF-β and IGF-I in disc. Apoptosis in the disc was evaluated using TUNEL staining. Results: Coculturing MenSCs with AF cells demonstrated their ability to express collagen I and biomarkers of AF cells. Moreover, the coculture system led to upregulation of the growth factors TGF-β and IGF-I. After 12 weeks, discs treated with MenSCs gel exhibited significantly lower Pffirrmann scores (2.29 ± 0.18), compared to discs treated with MenSCs (3.43 ± 0.37, p < 0.05) or gel (3.71 ± 0.29, p < 0.01) alone. There is significant higher MR index in disc treated with MenSCs gel than that treated with MenSCs (0.51 ± 0.05 vs 0.24 ± 0.04, p < 0.01) or gel (0.51 ± 0.05 vs 0.26 ± 0.06, p < 0.01) alone. Additionally, MenSCs gel demonstrated preservation of the structure of degenerated discs as indicated by histological scoring (5.43 ± 0.43 vs 9.71 ± 1.04 in MenSCs group and 10.86 ± 0.63 in gel group, both p < 0.01), increased aggrecan expression, and decreased MMP13 expression in vivo. Furthermore, the percentage of TUNEL-positive cells in the disc treated with MenSCs Gel (26.59%) was significantly lower than those treated with gel alone (55.77%, p < 0.01) and MenSCs alone (50.95%, p < 0.01). The expression of TGF-β and IGF-I was higher in discs treated with MenSCs gel or MenSCs alone than in those treated with gel alone. Conclusion: MenSCs embedded in collagen I gel has the potential to preserve the disc structure and prevent disc degeneration after discectomy, which was probably attributed to the paracrine of growth factors of MenSCs.