In an attempt to find an ideal closure method during annulus defect repair, we evaluate the use of medical glue by mechanical and biocompatible test. Cyanoacrylate medical glue was applied together with a multilayer microfiber/nanofiber polycaprolactone scaffold and suture in annulus repair. Continuous axial loading and fatigue mechanical test was performed. Furthermore, the in vitro response of mesenchymal stem cell (MSC) to the glue was evaluated by cell viability assay. The in vivo response of annulus tissue to the glue and scaffold was also studied in porcine lumbar spine; histological sections were evaluated after 3 months. Cyanoacrylate glue significantly improved the closure effect in the experimental group with failure load 2825.7 ± 941.6 N, compared to 774.1 ± 281.3 N in the control group without glue application (p < 0.01). The experimental group also withstood the fatigue test. No toxic effect was observed by in vitro cell culture and in vivo implantation. On the basis of this initial evaluation, the use of cyanoacrylate medical glue improves closure effect with no toxicity in annulus defect repair. This method of annulus repair merits further effectiveness study in vivo. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015.