Objective: To investigate the effect and mechanism of miRNA-326 overexpression on apoptosis of degenerative nucleus pulposus (NP) cells in intervertebral disc. Methods: MiRNA-326 lentivirus expression vector was constructed and recombinant lentivirus was obtained in 293T cells. After infection with NP cells, the stable overexpressed cell line GV369-miRNA-326-NP was obtained, at the same time, the empty vector GV369-NP group and the blank group were set up. The expression of lentivirus label protein GFP was observed by fluorescence microscope. The expression of miRNA-326 was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Apoptosis of cells was detected by flow cytometry. The targeting relationship between miRNA-326 and FasL was verified by Luciferase reporter gene analysis and the expression of apoptosis-related proteins (FADD, caspase-3, Bcl-2 and Bax) was detected by Western bloting. The change of cell mitochondrial membrane potential was detected with the kit. Results: Under fluorescence microscope, green fluorescence was observed in the lentivirus infected overexpressed cell lines and empty carrier cell lines, but not in the blank cell group. Compared with the blank group, the expression levels of miRNA-326, Bcl-2 and mitochondrial membrane potential in the GV369-miRNA-326-NP group were significantly increased, while the expression levels of apoptosis rate, FADD, caspase-3 and Bax were significantly decreased, with statistically significant differences (P<0.05). There was no statistically significant difference between the GV369-NP group and the blank group (P>0.05). Luciferase reporter gene analysis confirmed a targeted relationship between miRNA-326 and FasL. Conclusion: MiRNA-326 can inhibit the apoptosis of degenerative nucleus pulposus cells in intervertebral disc. It can not only participate in the apoptosis mediated by caspase-3 and FADD through the targeted regulation of exogenous FasL/Fas pathway, but also play a role in the apoptosis through mitochondrial pathway.